ACE2 Expression in Symptomatic COVID-19 Patients
ACE2 Expression in Symptomatic COVID-19 Patients

ACE2 Expression in Symptomatic COVID-19 Patients - PowerPoint Presentation

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ACE2 Expression in Symptomatic COVID-19 Patients - PPT Presentation

Mikki Jaramillo Indiana University School of Dentistry Mythily Srinivasan BDS MDS PhD Indiana University School of Dentistry Thankam Thyvalikakath DMD BDS MDS PhD Indiana University School of Dentistry ID: 1000289

cov2 sars saliva covid sars cov2 covid saliva samples sace2 ace2 viral uws patients assay symptomatic sample cells indiana




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1. ACE2 Expression in Symptomatic COVID-19 PatientsMikki Jaramillo, Indiana University School of Dentistry Mythily Srinivasan, BDS, MDS, PhD, Indiana University School of DentistryThankam Thyvalikakath, DMD, BDS, MDS, PhD, Indiana University School of Dentistry ContactMikki JaramilloIndiana University School of Dentistrymikjaram@iu.eduReferences[1] Srinivasan, M., Zunt, S. L., & Goldblatt, L. I. (2020). Oral epithelial expression of angiotensin converting enzyme-2: Implications for COVID-19 diagnosis and prognosis. BioRxiv. https://doi.org/10.1101/2020.06.22.165035[2] Srinivasan, M., Thyvalikakath, T. P., Cook, B. N., & Zero, D. T. (2020). COVID-19 and saliva: A primer for dental health care professionals. International dental journal, 10.1111/idj.12606. Advance online publication. https://doi.org/10.1111/idj.12606[3] Rahman, M. M., Hasan, M., & Ahmed, A. (2021). Potential detrimental role of soluble ACE2 in severe COVID-19 comorbid patients. Reviews in medical virology, 10.1002/rmv.2213. Advance online publication. https://doi.org/10.1002/rmv.2213 The cycle threshold (Ct) was determined by the number of PCR cycles necessary to produce a fluorescence above that of the background in the Abbott SARS-CoV2 assay. The COVID-19 UWS samples had Ct values ranging from 13.60 to 30.47 (Fig 2B). The viral load in each UWS was determined from the Ct value by following the manufacturer’s manual. The SARS-CoV2 viral load in the COVID-19 UWS samples ranged from 1.6 to 678 copies per mL (Fig 2C). A standard curve was created by plotting OD values versus the amount of ACE2 Assay Standard. A linear regression was applied to determine the slope values at each data point. The binding activity was calculated according to the following equation where OD is optical density, Blank is the negative control, and Slope is calculated from the linear regression (Fig 3). sACE2 was significantly lower in COVID-19 saliva samples as compared to the healthy UWS. Methods Study cohort: Unstimulated whole saliva (UWS) was collected from symptomatic COVID-19 patients diagnosed by the Abbott SARS-CoV2 assay using nasopharyngeal swabs and admitted to the Indiana University hospital system, with the help of the Indiana Biobank in accordance with their institutional review board. Control UWS included archived saliva samples from the salivary research laboratory at Indiana University School of Dentistry. Virus detection: SARS-CoV2 in UWS was detected using Abbott’s kit. This assay uses a reverse transcriptase PCR reaction to target the RdRp and N genes of SARS-CoV2. Measurement of sACE2 in saliva: The UWS samples were analyzed for sACE2 using the CoviDrop SARS-CoV2 Spike-ACE2 Binding ELISA assay following manufacturer’s instruction (Epigentek). Statistical analysis: Differences between the COVID-19 and healthy samples were assessed by the student’s t-test. Of the samples from symptomatic COVID-19 patients, five were discarded due to insufficient volume, seven were negative for SARS-CoV2, and two were inconclusive as determined by the Abbott SARS-CoV2 assay. The remaining 16 samples were positive (Fig 2A). The discrepancy between the nasopharyngeal swab results and the saliva test for detecting SARS-CoV2 could be attributed to any of the following: 1) the time of sample collection with respect to the course of infection, 2) the nature of the sample, and 3) the differences in the sensitivity between nasopharyngeal swab and saliva samples. Typically, saliva was collected approximately 24-48 hours after nasopharyngeal swab, independent of the patient’s course of infection. The nature of saliva samples may vary between patients in terms of potential inhibitors and quenchers. The COVID-19 positive saliva samples exhibited significantly lower sACE2 as compared with the saliva samples from healthy individuals. There was a weak correlation between the viral load and the sACE2 in UWS in symptomatic COVID-19, which could be attributed to the small sample cohort. Results Discussion Conclusion Our data suggest that sACE2 in UWS could represent a marker for symptomatic COVID-19. However, assessment of sACE2 in a larger population and asymptomatic COVID-19 patients needs to be established to validate. Introduction The novel coronavirus SARS-CoV2 first emerged in December 2019. Since then, it has quickly spread to a pandemic health crisis, causing the multisystem disease COVID-19. As of March 2021, the United States has had over 29 million COVID-19 infections and over 545,000 COVID-19 related deaths. SARS-CoV2 is primarily transmitted person-to-person through airborne particles and through direct and indirect contact of mucosal surfaces. Common symptoms of COVID-19 include fever, cough, and dyspnea. This can progress into respiratory distress, pneumonia, and death. Oral symptoms, such as mucosal lesions and dysgeusia, are often early manifestations. To enter the host cells, SARS-CoV2 binds via the S1 subunit of the spike protein to a virus binding motif on the angiotensin converting enzyme-2 (ACE2) on the host cells (Fig 1). Then, the transmembrane serine protease 2 on the surface of endothelial cells cleaves the spike protein between the S1 and S2 subunits, allowing the virus to fuse with the cell. Following viral entry, the metalloprotease ADAM-17 cleaves the extracellular domain, resulting in the release of a soluble form of ACE2 (sACE2). ACE2 functions as a counterbalance to the effects of ACE in the RAAS pathway, reducing blood pressure and inflammation. ACE2 is expressed in the epithelial cells of the lung alveoli, intestinal lining, oral mucosa, and salivary glands. The sACE2 form can bind to the circulating SARS-CoV2 and sequester the circulating SARS-CoV2. Alternatively, coating with sACE2 could facilitate the viral entry into the host at distant sites and thus play a role in disease expression. The objective of this project was to evaluate the presence of sACE2 in saliva of healthy and symptomatic SARS-CoV2 infected individuals. Figure 1: ACE2 expression and SARS-CoV2 pathophysiology. (A) ACE2 is present in epithelial cells of oral mucosa. (B) The spike protein of SARS-CoV2 binds to ACE2. Subsequent cleaved of the S1 and S2 subunits on the spike protein allow the viral particle to enter the host. (C) ACE2 on exfoliated oral epithelial cells and released sACE2 are present in saliva.[1] Figure 2: Detection of SARS-CoV2 in saliva samples. (A) The sample cohort included 30 samples from patients hospitalized with COVID-19 who tested positive for SARS-CoV2 by nasopharyngeal swabs. (B) Mean Ct value from Abbott SARS-CoV2 assay ranged between 13.60 and 30.47 in 16 saliva samples and were diagnosed as positive for infection with SARS-CoV2. (C) The SARS-CoV2 viral load for samples 2-12, 14, and 15 ranged from 1.6 to 109 viral copies per mL. The SARS-CoV2 viral load for sample 13 was 678 viral copies per mL. Figure 3: ACE2 is decreased in saliva of COVID-19 patients. The sACE2 saliva level is determined by its ability to bind to a plate pre-coated with the SARS-CoV2 spike protein in an ELISA assay. Saliva of symptomatic COVID-19 patients exhibited significantly lower sACE2 as compared to that in the saliva of healthy individuals with a p-value less than 0.05. *=p<0.05 A) Sample CohortB) Mean Ct Values from Abbott SARS-CoV2 AssayC) SARS-CoV2 Viral LoadSample 13*Binding Activity