3.1 Discovery of broad oral infection by SARS-CoV-2
Despite the potential for direct oral tissue infection by SARS-CoV-2 and the oral symptoms in acute COVID-19 (Table 1), the challenge has been to distinguish causation from correlation. Critical questions related remained as to whether the various oral symptoms were because of infection oral tissues by SARS-CoV-2 or if these COVID-19 manifestations were related to generalized, severe inflammation from an infection elsewhere in the body. Additionally, many groups were interested in the potential infectiousness of saliva (Figure 1C). The resolution of these questions required extensive collaboration between oral health care scientists and providers, medical doctors, basic scientists, and bioinformaticians. The implications for comparing oral and nasopharyngeal viral load concordance12 as well as discovering primary oral infection by SARS-CoV-2 are relevant for testing in the pre-symptomatic stage, identifying symptomatic infection, and providing post-acute COVID-19 patient care (Figure 2).
|Taste loss or alterations (ageusia, hypogeusia, dysgeusia)||Tongue ****|
|Papillitis, glossitis (also known as “COVID tongue”)||Tongue ***|
|White-coated tongue||Tongue *|
|Mucositis, pinpoint ulcerations, erythema||Oral mucosa **|
|Mucosal plaques||Oral mucosa *|
|Mucosal desquamation||Oral mucosa **|
|Enanthema, petechiae||Oral mucosa *|
|Pustules, blisters, vesicles, macula, hyperpigmentation||Oral mucosa *|
|Aphthous stomatitis||Oral mucosa *|
|Necrotizing periodontal disease||Periodontium *|
|Parotitis or sialadenitis||Salivary glands ****|
|Xerostomia or hyposalivation||Salivary glands ****|
|Trigeminal neuralgia||Neurologic *|
|Facial tingling||Neurologic *|
|Temporomandibular joint abnormalities||Neurologic *|
|Candidiasis (also known as thrush)||Generalized *|
|Burning mouth or burning, itching sensations (pruritus)||Generalized *|
The highlighted findings and new data presented here build off incredible efforts from the international research community and funding organizations who supported the rapid study of SARS-CoV-2 since December 2019 (Figure 2). The dissemination of COVID-19 discoveries by scientists to other scientists and the public has been dynamic, and synthesizing relevant evidence requires listening to patient experiences, reading news sources, and collecting reports across hundreds of publications—including the exponential increase of preprints on bioRxiv and medRxiv. Given the urgent need to answer questions, the availability of preprint articles was critical to driving hypotheses and collaborations—with some appearing nearly a year before official publication in a peer-reviewed journal.13 Multiple key findings were rapidly released and changed clinical recommendations (see: The “Oral” History of COVID-19 Timeline in Figure 3).
An example of an oral finding that was shared rapidly and was critically important in driving healthcare decisions is represented by the rapid publication following anecdotal reports in March 2020 that health care providers recommended that patients stay home if they experienced a loss of taste or smell. The first report was published on this phenomenon as a preprint publication on March 26.14 This prompted the American Academy of Otolaryngology in late March to propose adding smell and taste alterations to COVID-19 screening protocols. This update to the screening protocols turned out to be critical: The documented prevalence of taste alterations is now reported in nearly 50% of all COVID-19 patients.15 Sudden changes to olfaction and gustation are more prevalent in this disease compared to other viral diseases and now serve as a key COVID-19 symptom.16
Data indicating that the oral cavity could be a site of SARS-CoV-2 infection began to accumulate (Figure 3). The first case reports of oral mucosal lesions were published in late April 2020.17 A few weeks later, a study published using the COVID Symptom Study smartphone-based app compiled data from 2,618,862 participants, and 65% of respondents reported taste loss.18 Further, this project demonstrated the future potential for crowdsourcing disease data in real-time. One-off reports were also published regarding high transmission rates from activities that involve the oral cavity such as singing in choir practice.19 At the same time, others published hypotheses that the oral cavity may be a site of direct infection in COVID-1920 or that periodontal disease may serve as a risk factor for severe COVID-19.21 By summer 2020, other less common oral symptoms were being reported more often in COVID-19 patients, including salivary gland inflammation22 and oral mucosal lesions23 (Table 1). Despite the numerous lines of evidence in support of an oral-COVID-19 axis—including a case report that detected the virus in an oral cancer resection24—it remained unclear whether these oral manifestations were evidence of primary infection of the oral cavity by SARS-CoV-2 or secondary to COVID-19-related inflammation. We also note that these are not mutually exclusive and that some pharmacotherapies for COVID-19 may also contribute to these reported oral signs and symptoms.
Data were needed to confirm that each reported oral manifestation results from SARS-CoV-2 infection, including the analysis of the viral entry factors in each oral tissue niche. Additionally, identification of infection in situ is critical. By early February 2020, one receptor for SARS-CoV-2 cellular entry was known: angiotensin-converting enzyme 2 (ACE2).25 This rapid discovery was possible because previous researchers had determined that SARS-CoV, the causative agent of the 2003 SARS pandemic, used ACE2 to enter cells.26 Weeks later, Xu et al. published a single-cell transcriptomic analysis of ACE2 expression in the human tongue, gingiva, and buccal mucosal cells that suggested the tongue was especially at risk for direct infection by SARS-CoV-2.27 Although these studies provided evidence for oral mucosal risk for infection the salivary glands were not included in this study. Subsequent reports suggested that the salivary glands were similarly at risk and, if infected, may serve as a primary cause of asymptomatic transmission.28 Based on these early studies and the heterogeneity of oral epithelial cells in distinct niches,29 we hypothesized some oral cell types in the mucosa and the glands would be especially susceptible to infection.
Most groups studying SARS-CoV-2 host entry factors focused on ACE2, the TMPRSS protease family (TMPRSS2, TMPRRS4, and TMPRSS11D), and cathepsins (CTSB and CSTL).30 The power of collaboration was evident in the multiple published studies by groups associated with the Human Cell Atlas (HCA). These international collaborations integrated single-cell transcriptomic assays from multiple tissues to identify cell-specific vulnerabilities for infection by SARS-CoV-2 throughout the body,31 resulting in the COVID-19 Cell Atlas (https://www.covid19cellatlas.org/). As part of this effort, our team donated single cell RNA sequencing datasets of human gingiva and salivary glands to this atlas in June 2020, leading to the creation of the Oral & Craniofacial Biological Network (HCA-OCBN) in September 2020. Using our group’s single cell atlases, we found broad expression of ACE2 and genes encoding TMPRSS family members in barrier epithelial subpopulations.32 Here, we show the results of the examination of the sex-specific expression of these viral entry host genes (Figure 4A). Our results indicate that the expression of these entry factor-encoding genes was similar in epithelial cells from males and females, despite the higher burden of acute COVID-19 in older males with defined comorbidities.33 Whether the oral host response to the viral infection is distinct between sex is still unexplored.
For low abundance transcripts like those for ACE2, single-cell transcriptomics analyses often serve as a relative guide rather than an absolute measurement. We estimate that low abundance transcripts could be underestimated by a range of 1 in 5 to 10 transcripts per cell (unpublished observations). To evaluate specific entry factors with greater sensitivity, we designed probes to map these entry factor transcripts throughout the oral epithelia sites using multiple techniques including in situ hybridization.32 Based on these analyses,32 we conclude that (1) all oral mucosae and glands are at risk for infection by SARS-CoV-2, and (2) the most at-risk epithelial cells are those exposed to the external environment. These highly at-risk epithelial cells include the gingiva, where ACE2, TMPRSS2, and TMPRSS4 were enriched in the sulcular and junctional epithelia (Figure 4B) and are the same cells that constantly shed into saliva and may stick to medical gloves when performing an oral exam (Figure 4C). To determine if these sites were infected, we obtained specimens from the oral cavity from the NIH COVID-19 Autopsy Consortium. Using multiple assays, we found infection and viral replication of the major and minor salivary glands and oral mucosae (Figure 4C, D).32
Finding viral entry factors, infection, and viral replication within multiple tissues of the oral cavity provides strong evidence that some of the reported oral COVID-19 manifestations are likely because of direct infection by SARS-CoV-2. Many of the oral COVID-19 manifestations relate to inflammation (Table 1). Indeed, tissue damage and cell death, mediated by T cells and B cells, occurs in response to SARS-CoV-2 infection.34 Here, we show data of the local oral immune response to salivary gland infections by SARS-CoV-2 (Figure 4D). The infected glands exhibited pathologic signs of sialadenitis (salivary gland inflammation) that was associated with T cell infiltration (see Huang et al.32). Though we found oral mucosal infection of a non-keratinized site, the salivary glands are the only confirmed oral niche to date, that has been proven to be the result of primary infection. To do this required discovery that 1) the glands and mucosa express genes encoding host factors that mediate viral entry and 2) that these sites were confirmed to be a site of SARS-CoV-2 infection and replication. However, only the glands were the only sites identified and characterized by a robust host immune response at the site of infection.
These findings underscore the importance of further research to understand the consequences of SARS-CoV-2 infection in other oral niches. For example, the first report of direct infection of the taste papillae was just published, suggesting a substantial effect on the epithelial progenitor population.35 Determining correlation or causation for other oral manifestations still requires an organized and motivated research community to share research and resources. Open questions include how these oral niche susceptibilities change during childhood development into adulthood, how chronic oral inflammatory conditions influence viral infection and systemic disease progression, which of the reported oral conditions are a direct result of viral infection of the oral tissues, and in those infected sites, what impact SARS-CoV-2 infection had on those tissues long-term.
3.2 Salivary transmission, antibody response, and predictive potential
In April 2020, one of the first COVID-19 clinical trials (NCT04348240) at the NIH was started. This trial aimed to evaluate the transmissibility and viral load of SARS-CoV-2 from saliva and the nasopharynx.32 This study was also part of our oral-COVID-19 work, recruiting acute COVID-19 and at-risk cohabitating subjects and monitoring them both prospectively. We found that saliva from individuals with mild COVID-19 or even asymptomatic individuals contained substantial concentrations of free virus mixed with infected cells shed from the oral mucosae, suggesting that the oral cavity alone may be a source of viral transmissibility within and between individuals. This saliva also was monitored prospectively for IgG antibodies, with the results closely matching blood antibody profiles and levels, suggesting the potential for neutralization against SARS-CoV-2 in most subjects by 3 weeks post-diagnosis. These results further demonstrate the potential for non-invasive oral sampling as a method for studying systemic immunity in COVID-19 and for testing for SARS-CoV-2 infection.32
Additionally, a cohort of NIH employee saliva samples was studied, revealing that some individuals who remained asymptomatic could display high saliva viral loads (measured by CT values as high as < 20). These values could be higher than symptomatic patients. The saliva from these asymptomatic individuals was discovered to contain both free virus and the SARS-CoV-2-infected epithelial cells; both were capable of transmitting SARS-CoV-2 in culture. Overall, these preliminary data show that the oral cavity represents a robust and underappreciated site for SARS-CoV-2 infection and raises the possibility that the oral cavity actively participates in viral transmission, even in asymptomatic individuals.
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