Detection of SARS‐CoV‐2 in saliva and characterization of oral symptoms in COVID‐19 patients

Abstract Objectives In order to provide a more comprehensive understanding of the effects of SARS‐CoV‐2 on oral health and possible saliva transmission, we performed RNA‐seq profiles analysis from public databases and also a questionnaire survey on oral‐related symptoms of COVID‐19 patients. Materials and methods To analyse ACE2 expression in salivary glands, bulk RNA‐seq profiles from four public datasets including 31 COVID‐19 patients were recruited. Saliva and oropharyngeal swabs were collected. SARS‐CoV‐2 nucleic acids in saliva were detected by real‐time polymerase chain reaction (RT‐PCR). Additionally, a questionnaire survey on various oral symptoms such as dry mouth and amblygeustia was also carried out on COVID‐19 patients. Results ACE2 expression was present at detectable levels in the salivary glands. In addition, of four cases with positive detection of salivary SARS‐CoV‐2 nucleic acids, three (75%) were critically ill on ventilator support. Furthermore, we observed the two major oral‐related symptoms, dry mouth (46.3%) and amblygeustia (47.2%), were manifested by a relatively high proportion of 108 COVID‐19 patients who accepted the questionnaire survey. Conclusions This study confirms the expression of ACE2 in the salivary glands and demonstrates the possibility of SARS‐CoV‐2 infection of salivary glands. Saliva may be a new source of diagnostic specimens for critically ill patients, since it can be easily collected without any invasive procedures. In addition, dry mouth and amblygeustia can be considered as initial symptoms of COVID‐19 infection.


| INTRODUC TI ON
Since December 2019, an outbreak of a novel coronavirus (SARS-CoV-2), which started in Wuhan, China, has been spreading rapidly, resulting in a potentially life-threatening viral respiratory disease named as coronavirus disease  by the World Health Organization (WHO). 1 By 23 August 2020, more than 20 million cases of COVID-19 and more than 800 000 deaths have been confirmed in over 200 countries, which has been classified as a pandemic by the WHO on 11 March 2020. 2,3 This in turn had a severely detrimental impact on the public health and economy of several Like blood, saliva is rich in multiple biological markers such as DNA, RNA, proteins, with readily detectable levels of microorganism, as the two biofluids share many similarities in molecular components. 5,6 Hence, there is a high probability of antibodies and viruses of the human body being present in saliva. It has been reported that some viruses of large-scale infectious diseases, particularly the respiratory diseases such as severe acute respiratory syndrome (SARS) and middle east respiratory syndrome (MERS), can be detected in saliva. 7,8 Moreover, saliva has already been utilized for the detection of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and various drugs like cocaine and alcohol. 9,10 As a result, it is imperative to determine whether SARS-CoV-2 can be detected in saliva, as well as characterize the initial oral symptoms manifested in COVID-19 patients.
Single-cell RNA-seq data analysis of angiotensin-converting enzyme II (ACE2) expression and serological investigation of patient samples have indicated that ACE2 may be the cell receptor of SARS-CoV-2, 11,12 thus suggesting that ACE2-expressing cells are likely to be the major target cell type which are vulnerable to SARS-CoV-2 infection. There is typically high expression of ACE2 r on the epithelial cells of oral mucosa, which is particularly enriched in the epithelial cells of the tongue. 13 Prior to this study, there have been few reports that specifically investigated whether the salivary gland epithelial cells express ACE2 receptors. To confirm this, bulk RNA-seq profiles from four public datasets (https://www.prote inatl as.org/) including GTEx dataset, HPA dataset, FANTOM5 dataset and Consensus dataset were analysed. Furthermore, to verify that there are detectable levels of SARS-CoV-2 nucleic acids in saliva, we have screened the saliva of COVID-19 patients (who displayed positive results for SARS-CoV-2 nucleic acids detection before or on the day of sample collection). The results have been compared with oropharyngeal swabs, which is one of three diagnostic criteria. 4 Additionally, we also carried out a survey on the oral health status of COVID-19 patients, to evaluate the health status of the salivary glands. Our study thus provides a more comprehensive understanding of the detection of SARS-CoV-2 in saliva, and initial oral symptoms upon COVID-19 infection of oral tissues.

| Public datasets acquisition
The public bulk RNA-seq profiles were collected from the Human Protein Altas (available from https://www.prote inatl as.org/) including four datasets (GTEx dataset, HPA dataset, FANTOM5 dataset and Consensus dataset). GTEx data and HPA data were showed as mean pTPM (protein-coding transcripts per million). FANTOM5 data were obtained through Cap Analysis of Gene Expression (CAGE), which were reported as Scaled Tags Per Million. Data from Consensus were combining the data from GTEx, HPA and FANTOM5 datasets using the internal normalization pipeline, which were reported as Normalized eXpression (NX) levels. Our main focus is on the tissue distribution and expression of ACE2, particularly in the salivary gland.

| Inclusion criteria for patients
Inclusion of patients in this study is in accordance with (a) the diagnostic guidelines for new coronavirus pneumonia (NCIP) of the seventh edition, 4 and (b) SARS-CoV-2 nucleic acids detection remaining positive before or on the day of sample collection in our study. The inclusion criteria were set to ensure that the patients we selected in our study were tested positive for SARS-CoV-2 nucleic acids before collection of the saliva samples. According to the inclusion criteria described above, 31 patients were selected. be a new source of diagnostic specimens for critically ill patients, since it can be easily collected without any invasive procedures. In addition, dry mouth and amblygeustia can be considered as initial symptoms of COVID-19 infection.
According to NCIP of the seventh edition, 26 patients were of the ordinary or heavy type, and 5 patients on ventilator support were of the critically ill type.

| Saliva collection
After cessation of eating or drinking for 30 minutes, the oral cavity of each patient was cleaned by normal saline before saliva collection.
Then the tongue of each patient was lifted to expose the opening of the salivary gland duct. Upon gentle massage of the salivary gland, pure saliva was secreted. In this study, we gently collected about 1.5 mL of midstream salivary fluid with cotton swabs. The samples were placed into sterile dry containers immediately, without touching the inside of the cap or inner walls before closing the cap.

| Oropharyngeal swab collection
Oropharyngeal swabs and saliva samples were collected at the same time. A synthetic fibre swab was inserted into the patient's throat from the mouth and the posterior pharynx was swabbed, avoiding the tongue. After swabbing, each absorbent swab was placed immediately into a sterile tube.

| Nucleic acids extraction and real-time polymerase chain reaction (RT-PCR) of SARS-CoV-2
According to the latest guidelines issued by WHO, nucleic acids molecular testing of the virus is essential for diagnosis, 14 with the genetic targets in China being ORF1ab and nCoV-N. 15 The SARS-CoV-2 was detected by using a RT-PCR system by following the commercial test kit instructions (BioGerm. InC). The test results were obtained on a Roche Cobas z480 PCR Analyzer. A CT value of less than 35 (or 35 < CT<38 for twice) was defined as positive. Primer sequences used for amplification are listed in Table 1.

| RE SULTS
The data from GTEx dataset, HPA dataset, FANTOM5 dataset, and Consensus dataset in the Human Protein Atlas (Human Protein Atlas available from https://www.prote inatl as.org/, images/data available from v19.3.prote inatl as.org) showed the expression and distribution of ACE2 within normal human tissues, particularly in salivary glands.
The sample ID in GTEx data set, HPA data set and FANTOM5 data set is shown in Tables S1-S3. The results showed that ACE2 is highly expressed in the gastrointestinal system, testes, kidney and heart muscles. Although ACE2 is also expressed in the salivary glands, its expression is at a relatively low level. The results of RNA-seq showed that in normal salivary gland tissues, ACE2 is mainly expressed by glandular cells (Figure 1).
In 31 COVID-19 patients, based on the inclusion criteria, there were 15 male and 16 female patients. The median age was 60.6 years, ranging from 18 to 86 years. There were 5 critically ill patients who needed ventilator support to breathe in these 31 cases. Saliva and oropharyngeal swabs samples were collected at the same time. In our study, 13 cases were tested positive for oropharyngeal swab nucleic acids detection. Among these 13 patients, there were 4 cases with positive nucleic acids detection in saliva, of which 3 cases were critically ill patients on ventilator support, and 1 case being ordinary or heavy without ventilator support ( Table 2). The information and test results of the 4 cases are shown in Table 3.
Of the 108 valid questionnaires, 52 respondents were male and 56 respondents were female. The age data of one female patient were missing. After excluding this case, the overall average age was 52.0, 51.1 years for males and 52.9 years for females.
Among the 14 oral-related symptoms listed, amblygeustia (47.2% overall, 36.5% in males, 57.1% in females) and dry mouth (46.3% overall, 46.2% in males, 46.4% in females) had the highest incidence. The ACE2 expression was present at detectable levels in the salivary glands, but was lower than other tissues like the gastrointestinal system, testes, kidney and heart muscles ( Figure 1). This might explain why there was just one positive salivary detection in ordinary and heavy patients. A previous study reported the detection rate of SARS-CoV-2 in self-collected saliva. 16 However, in that study, patients were asked to cough out saliva from their throat into sterile containers, and hence the saliva samples were mainly sputum from the lower respiratory tract. In our study, the saliva samples were the pure fluid from the canal of salivary glands.
It is noteworthy that the positive saliva detection rate was as Another result worth noting in this study is that according to the analysis of the questionnaire, the two major oral-related symptoms, dry mouth and amblygeustia, were manifested by a relatively high proportion of 108 COVID-19 patients. A previous study identified fever, fatigue and dry cough as the most common symptoms of COVID-19 infection. 17 However, a number of recent studies have found that COVID-19 patients generally have oral symptoms such as dry mouth and amblygeustia, which are described in a review by Pellegrino et al. 18 An international multicentre study collected information from 394 patients diagnosed with COVID-19 from multiple countries, 161 of whom had olfactory or taste disorders, accounting for 41%. 19 A cross-sectional study reported that 20% of COVID-19 patients would feel taste abnormalities. 20 There have been news reports that even children recovering from mild COVID-19 are still unable to taste food. 21 The researchers found a correlation between dry mouth and bitter taste in COVID-19 patients in a recently published study. 22 Ren et al also suggest that COVID-19 patients may develop early oral symptoms (including taste loss and dry mouth), even before fever and dry cough. 23 Therefore, some experts have suggested that olfactory or taste disorders should be included in COVID-19 screening criteria. 24 Oral health researchers may play a more active role in the early diagnosis and treatment of COVID-19 by exploring the mechanism of dry mouth and amblygeustia.
Besides viral invasion, we speculate that oral symptoms may also be due to the patient's change in psychological status, poor oral hygiene or microbiota imbalance caused by therapeutic drugs. In particular, high ACE2 expression can be found on the epithelial cells of oral mucosa, which is enriched in epithelial cells of the tongue, thus providing possible routes of entry for SARS-CoV-2. 13 Therefore, oral mucosa might be at potential risk of infection by SARS-CoV-2, suggesting that oral symptoms could also be considered as initial symp- This study thus confirms that ACE2 is also expressed in the salivary glands, SARS-CoV-2 can be detected in saliva, and oral symptoms may be frequently manifested by COVID-19 patients.
The findings of this study suggest that saliva may carry a risk of SARS-CoV-2 transmission, particularly in critically ill patients, and that SARS-CoV-2 could cause partial impairment of oral tissues, thus providing a new insight to the clinical prevention, diagnosis and treatment of COVID-19. More in vitro and in vivo evidence and indepth histological data are needed to further confirm and reinforce these findings.

CO N FLI C T O F I NTE R E S T
We declare no competing interests.

AUTH O R CO NTR I B UTI O N S
S. W., L. W. and Y. J. designed and supervised this study, and revised the manuscript; L. C. carried out the experiments, analysed the data, wrote and revised the article; J. Z. and J. P. searched the databases, analysed the data, wrote and revised the article; X. D. analysed the data and revised the article; X. L., Z. S. and F. G. collected clinical samples and case data; Z. G. and Q. Z. performed the laboratory testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.