key: cord-0846764-2jyp3ift
authors: Chaudhary, Prem Prashant; Melkonyan, Arsen; Meethil, Archana; Saraswat, Shweta; Hall, David L.; Cottle, James; Wenzel, Mark; Ayouty, Nadine; Bense, Spenser; Casanova, Fabiola; Chaney, Matthew; Chase, Hannah; Hermel, Rebecca; McClement, Matthew; Sesson, Claire; Woolsey, Bryce; Kumar, Purnima
title: Estimating salivary carriage of SARS-CoV2 in non-symptomatic individuals and efficacy of mouthwash in reducing viral load: a randomized controlled trial
date: 2021-06-11
journal: J Am Dent Assoc
DOI: 10.1016/j.adaj.2021.05.021
sha: a879073f290f46fcef99545a94d5abaf03e62a71
doc_id: 846764
cord_uid: 2jyp3ift

Background Many individuals infected SARS-COV2 never develop significant symptoms. With 24 million Americans already infected and highly transmissible variants rapidly emerging, it is highly probable that post- and pre-symptomatic individuals will form a significant fraction of those seeking dental care. Salivary carriage rates in these populations are not known. Moreover, although preventing transmission is critical for controlling spread, the efficacy of mouthrinses in reducing oral viral load is poorly studied.background Methods 201 asymptomatic, pre-symptomatic, post-symptomatic and symptomatic individuals were recruited and copy numbers of SARS-CoV2 measured in unstimulated saliva using real-time reverse transcriptase quantitative PCR. 41 symptomatic individuals were subsequently inducted into a randomized, triple-blinded study and instructed to rinse with saline, 1% hydrogen peroxide, 0.12% chlorhexidine or 0.5% povidone-iodine for sixty seconds. Viral load was measured 15- and 45-minutes post-rinsing. Results Salivary SARS-CoV2 was detected in 23% of asymptomatic, 60% of post-symptomatic and 28% of pre-symptomatic individuals. Neither carriage rate nor viral load correlated with COVID symptomatology, age, gender or ethnicity. All four mouthrinses decreased viral load by 61-89% at 15 minutes, and by 70-97% at 45 minutes. The extent of reduction correlated significantly with initial viral load.results Conclusions Non-symptomatic individuals can pose a risk for viral transmission, and mouthrinses are simple and efficacious means of reducing this risk, especially when the load is <104 copies/ml.conclusion Practical Implications At a time when resources are stretched, our findings contribute to evidence-based selection of personal protection equipment and simple infection control practices to reduce contagion at source.

On March 11, 2020, the World Health Organization declared COVID-19 as a pandemic.

On March 16, 2020, 198 ,000 dentists closed their doors to patients in the United States alone 1 .

Ten months later, restrictions on the use of certain instruments and key procedures are still in place 2 , fueling concern in the minds of providers and patients alike 3 , and leading to significant long-term changes in workflow patterns and re-configuration of operatories 4 . Regulators and health authorities based this guidance on studies that aerosols are potential vehicles for transmission of respiratory pathogens 5 and since SARS CoV-2 virus has been identified in saliva [6] [7] [8] , the potential for disease transmission through saliva has become a concern.

Pre-procedural mouthrinses have been used in dentistry for many years to reduce the microbial load in saliva [9] [10] [11] . Indeed, evidence from in vitro studies suggests that pre-procedural mouthrinses containing hydrogen peroxide or povidone iodine may help mitigate COVID-19 transmission 12, 13 , although the evidence from limited human studies is equivocal 14, 15 . Therefore, we aimed to examine the risk posed to dental personnel from potential patients who report no symptoms of COVID, and to investigate the efficacy of a simple intervention, that is, preprocedural mouth rinsing, on reduction in salivary viral load. We achieved this aim through a four-arm randomized controlled trial. To the best of our knowledge, this provides the first estimate of viral load in saliva of asymptomatic patients as well as an in vivo comparison of mouth rinses in COVID-19 patients.

The two arms of the study were approved by the institutional review board of for symptomatic group and (2) absence of any COVID-19 screening symptoms (based on the ADA questionnaire and body temperature) for the asymptomatic, pre-symptomatic and postsymptomatic groups. Exclusion criteria were: (1) allergy to any study mouth rinse, (2) active uncontrolled thyroid disease, (3) pregnancy and (4) patients undergoing radioactive iodine therapy. Subjects who were asymptomatic at initial presentation were followed up 48 and 72 hours later. Subjects who reported negative COVID symptoms at presentation, 48-and 72hours post-op were categorized as asymptomatic, those who were asymptomatic at initial presentation, but reported any of the symptoms listed in the ADA questionnaire at 48-or 72hours post-sampling were considered pre-symptomatic, and those who reported history of COVID (as confirmed by clinical symptoms and positive nasopharyngeal rt-qPCR test) were treated as post-symptomatic (Figure 1 ). Experimental Design: All subjects completed demographic, behavioral and health history questionnaires. Subjects were asked to collect saliva in their mouths for three minutes and then continuously drool into a tube containing RNA stabilizer 16 . Forty subjects being treated for COVID-19 were randomly assigned to receive a mouthrinse containing 15 milliliters of normal saline, 1% hydrogen peroxide, 0.12% chlorhexidine gluconate or 0.5% povidone-iodine. The primary outcome measure was reduction in viral load at 15 minutes. Based on previous J o u r n a l P r e -p r o o f studies 14, 15 , we estimated the reduction to range between 0 and 40%. Using an alpha of 0.05 to estimate effect size of 0.25 or less between groups, we estimated a group size of 10 using the DOE tool in JMP (SAS software). The mouthwash was dispensed in pre-measured quantities in colored bottles, and the individual dispensing the product was blinded to the type of mouthwash.

A block randomization protocol was used to create the randomization schedule in GraphPad (https://www.graphpad.com/quickcalcs/randomize2/). Subjects were also blinded to the identity of the mouthwash, to the extent that it was possible, given the variations in taste. Subjects vigorously rinsed with 7.5ml of the mouth rinse for 30 seconds, expectorated and rinsed with the remaining 7.5ml for a further 30 seconds. The two expectorants were collected and pooled. Statistical Analysis: Non-parametric comparisons were made between all pairs using Dunn's test for joint ranking, a method that incorporates Bonferroni adjustment for multiple tests. Chi-J o u r n a l P r e -p r o o f squared tests were used to evaluate differences in frequency of detection of COVID-19 between groups of subjects. Confidence intervals were calculated to confirm significant findings.

127 subjects were asymptomatic, 18 were pre-symptomatic and 41 were symptomatic and 15 were post-symptomatic. Post-symptomatic subjects reported 14-days or more of normal temperature.

SARS CoV-2 was detected in 23% of asymptomatic, 28% of pre-symptomatic, 99% of symptomatic individuals and 60% of post-symptomatic and individuals (Table 1) , however, the frequency of detection was significantly lower in asymptomatic and pre-symptomatic individuals when compared to symptomatic or post-symptomatic patients (p = 0.001, Chi-squared test). The viral load in saliva was also significantly lower in asymptomatic and pre-symptomatic individuals when compared to symptomatic or post-symptomatic patients (p = 0.0007, Dunn's test with joint ranking). In the symptomatic patients, those treated with remdesivir or convalescent plasma and/or demonstrable COVID-19 immunoglobulin titers tended to have lower salivary loads.

SARS-CoV-2 salivary carriage was greater in subjects who were early in the disease process.

These tendencies were not statistically significant. Salivary carriage of SARS-CoV-2 did not correlate with fever or loss of taste/smell in this group (p > 0.05, Chi-squared test).

No adverse events related to the mouthwash (pain/burning mouth, oral dryness, or difficulty in swallowing) were recorded during the trial. All four mouth rinses reduced salivary carriage of SARS-CoV-2 (Figure 2) . A median reduction of 61-89% (mean of 25-74%) was observed at 15 minutes, while the median reduction ranged from 70-97% at 45 minutes (mean of 30-43%). Neither the 15-miniute reduction in viral load, nor the persistence of reduction at 45 minutes differed between the mouthrinses (p>0.05, Dunn's test). There was a significant J o u r n a l P r e -p r o o f correlation between baseline viral load and reduction at 15 minutes (p = 0.0073, Spearman's rho), and persistence at 45 minutes (p=0.0087, Spearman's rho). In all individuals with a baseline viral load less than 10 4 copies/ml of saliva (n = 6), there was 100% reduction at 15 and 45 minutes.

The oral cavity forms a continuum with the nasopharynx and lower respiratory tract, and hence, aerosol generating dental procedures on patients with any type of infectious respiratory disease have the potential to create pathogen-rich aerosols; a concern that has gained immediacy in the SARS CoV2 pandemic. Moreover, oral epithelial cells, especially those in the tongue and saliva glands, express transmembrane protein angiotensin-converting enzyme (ACE2) and transmembrane serine protease 2 (TMPRSS2) 18, 19 , the primary receptors and portals of entrance of SARS-CoV-2 into human cells 20 . Since the oral cavity is a primary external interface, it is likely that these surfaces provide an avenue for viral colonization. This is borne out by our findings on asymptomatic, pre-symptomatic and post-symptomatic individuals (collectively referred to as non-symptomatic); and suggests that questionnaires based on disease symptomatology or temperature records might not be diagnostic of infective potential.

To the best of our knowledge, this is the first study to provide this information, and sets the stage for future work on risk of disease transmission among dental healthcare workers and patients.

Although we discovered that more than 40% of non-symptomatic individuals (including pre-, post-and asymptomatic subjects) carry the virus in their saliva, two lines of evidence point to a low risk for disease transmission from non-symptomatic patients. The first is based on mathematical modeling that contagion requires salivary viral loads of 10 8 copies/ml or greater 21 , J o u r n a l P r e -p r o o f and the clinical evidence that supports the model 22 . The second is from our earlier discovery that the primary source of aerosol microbiota is the irrigant that cools dental handpieces and ultrasonic scalers, and that SARS CoV-2 is not present in measurable levels in these aerosols when procedures are performed in conjunction with pre-operative mouthrinses and intra-oral high-volume evacuation (manuscript under review, JDR).

A recent survey reported that only 12% of American dentists currently administer preoperative mouthwashes 23 . In the present investigation, we demonstrate the efficacy of using any mouthwash, irrespective of its mechanism of action, on reducing salivary CoV-2 levels for up to 45 minutes. This efficacy directly correlated with salivary loads, further attesting to the benefits of pre-procedural mouth rinsing in non-symptomatic patients. Six subjects demonstrated a higher viral load at 45 minutes post-rinse when compared to baseline; in all of them, episodes of coughing were recorded during this period. COVID-19 viral loads in saliva including "deep cough sputum" are higher than nasopharyngeal loads at disease onset and decrease slower 24 .

It is possible that episodes of coughing might increase salivary viral load in non-symptomatic patients, and further studies are required to evaluate this.

In summary, within the limitations of a small sample size, we discovered a high rate of SARS CoV-2 carriage in the saliva of non-symptomatic individuals, although these levels are well below those required for disease transmission. We also discovered that mouthrinses are simple and highly efficacious means of reducing the virus from the oral environment for up to 45 minutes and may be a valuable tool in disease mitigation. 

Analysis of anxiety-related factors amongst frontline dental staff during the COVID-19 pandemic in Yichang, China

Dentistry during the COVID-19 Epidemic: An Italian Workflow for the Management of Dental Practice

Infection Prevention and Control Guidance for Dental Settings During the COVID-19 Response

SARS-CoV-2 prevalence in saliva and gastric and intestinal fluid in patients undergoing gastrointestinal endoscopy in COVID-19 endemic areas: prospective cross-sectional study in Japan

COVID-19 infection among emergency department healthcare providers in a large tertiary academic medical center following the peak of the pandemic

Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection

The effectiveness of a preprocedural mouthrinse containing cetylpyridinium chloride in reducing bacteria in the dental office

Chlorhexidine substantivity on salivary flora and plaque-like biofilm: an in situ model

Efficacy of Two Pre-Procedural Rinses at Two Different Temperatures in Reducing Aerosol Contamination Produced During Ultrasonic Scaling in a Dental Set-up -A Microbiological Study

In vitro virucidal activity of povidone iodine gargle and mouthwash against SARS-CoV-2: implications for dental practice

Differential effects of antiseptic mouth rinses on SARS-CoV-2 infectivity in vitro

A prospective clinical pilot study on the effects of a hydrogen peroxide mouthrinse on the intraoral viral load of SARS-CoV-2

Efficacy of commercial mouth-rinses on SARS-CoV-2 viral load in saliva: randomized control trial in Singapore

Methods for Collecting Saliva

SalivaDirect™: RNA extraction-free SARS-CoV-2 diagnostics. protocols

High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa

Oral epithelial expression of angiotensin converting enzyme-2: Implications for COVID-19 diagnosis and prognosis

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