key: cord-0965894-s86zb5up
authors: Carrouel, Florence; Valette, Martine; Gadea, Emilie; Esparcieux, Aurélie; Illes, Gabriela; Langlois, Marie Elodie; Perrier, Hervé; Dussart, Claude; Tramini, Paul; Ribaud, Mélina; Bouscambert-Duchamp, Maude; Bourgeois, Denis
title: Use of an antiviral mouthwash as a barrier measure in the sars-cov-2 transmission in adults with asymptomatic to mild COVID-19: a multicenter, randomized, double-blind controlled trial
date: 2021-05-24
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2021.05.028
sha: a6c9d3816cadbf54a037531acf1a0dba54cecb74
doc_id: 965894
cord_uid: s86zb5up

OBJECTIVES: To determine if commercially available mouthwash with ß-cyclodextrin and citrox (bioflavonoids) (CDCM) could decrease the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) salivary viral load. METHODS: In this RCT, SARS-CoV-2 PCR-positive patients aged 18-85 years with asymptomatic to mild COVID-19 symptoms <8 days were recruited. A total of 176 eligible patients were randomly assigned (1:1) to CDCM or placebo. Three rinses daily were performed for 7 days. Saliva sampling was performed on day 1 at 9 a.m. (T1), 1 p.m. (T2) and 6 p.m. (T3). On the following 6 days, one sample was taken at 3 p.m. Quantitative RT-PCR was used to detect SARS-CoV-2. RESULTS: The intention-to-treat analysis demonstrated that, over the course of one day, CDCM was significantly more effective than placebo 4 hours after the first dose (p=0.036), with a median percentage (log10 copies/mL) decrease T1-T2 of -12.58% [-29.55% - -0.16%]. The second dose maintained the low median value for the CDCM (3.08 log(10) copies/mL [0 - 4.19]), compared to placebo (3.31 [1.18 - 4.75]). At day 7, there was still a greater median percentage (log(10) copies/mL) decrease in salivary viral load over time in the CDCM group (-58.62% [-100% - -34.36%]) compared to placebo group (-50.62% [-100% - -27.66%]). These results were confirmed by the per-protocol analysis. CONCLUSIONS: This trial supports the relevance of using CDCM on day 1 (4 hours after the initial dose) to reduce the SARS-CoV-2 viral load in saliva. For long term effect (7 days), CDMC appears to provide a modest benefit compared to placebo in reducing viral load in saliva.

SARS-CoV-2 may be transmitted via saliva, even in patients who do not cough or have 57 other respiratory symptoms [1, 2] . SARS-CoV-2 is identified in 91.7% of saliva samples from 58 COVID-19 patients, and the load can reach up to 1.2×10 8 copies/mL [3] [4] [5] . When a person 59 sneezes, converses , breathes, or coughs, saliva droplets containing microorganisms are 60 produced [2] . The size of these droplets and the quantity produced depends on the individual. 61

Consequently, the risk of transmission is also variable [2] . The contamination between people 62 in close contact (1-3 m) occurs through saliva droplets (> 60 µm) [6] . The transmission 63 between two persons separated by a distance of up to 7 to 8 m is due to virus-laden aerosols 64 (droplets < 60 µm) [7, 8] . 65

The use of mouthwashes is an "adjuvant" treatment part of the usual treatment or 66 individual prophylaxis, especially in oral health. Considering mouthwashes as agents that can 67 decrease the viral load of SARS-CoV-2 is an extremely attractive concept [9-12]. However, 68 there is no in vivo evidence to recommend mouthwashes to control SARS-CoV-2 viral load. 69

Among antiviral molecules contained in mouthwashes, hydrogen peroxide, ß-cyclodextrin, 70 flavonoids, essential oils, cetylpyridinium chloride or povidone-iodine could be of interest in 71 the fight against SARS-CoV-2 [9, 10, 13] . The antiviral activity of CDCM in our trial is based 72 on ß-cyclodextrin and citrox (flavonoids). These molecules have demonstrated antiviral 73 activity against several viruses [14-20], but evidence for their action against SARS-CoV-2 74 was based only on in silico studies [10] . 75

The objective of our study was to describe the evolution of salivary SARS-CoV-2 76 viral load in COVID-19 outpatients receiving mouthwashes with or without antivirals. 77 Eligibility was restricted to adults aged 18-85 years old with a clinical diagnosis of 97 COVID-19 infection, asymptomatic or mild clinical symptoms that had been present for less 98 than 8 days, virological confirmation, an understanding and acceptance of the trial and written 99 agreement to participate in the trial. 100

The exclusion criteria were pregnancy, breastfeeding, an inability to comply with the 101 protocol, a lack of written agreement, mouthwash use on a regular basis (more than once a 102

week), an inability to answer questions and a lack of cooperation. 103 104

Eligible patients were randomly assigned (1:1) to either the ß-cyclodextrin-citrox 106 mouthwash (CDCM) group or the placebo group. The randomization sequence with 107 permutation blocks of size 4 was prepared using e-CRF Voozalyon 1.3 (Voozanoo, Caluire, 108 France) (Appendix p3). 109

Once enrolled, participants each received three 200 mL medication vials. Each vial 110 contained either a mouthwash containing the antiviral components (ß-cyclodextrin (0.1%) and 111 citrox (0.01%)) or placebo with similar appearance and content without the above-mentioned 112 antiviral components; the labels on the vials were identical. All participants, investigators, 113 statisticians, and laboratory staff were masked to medication vials and treatment allocation. Participants were instructed to use three mouthwashes per day (at 9 a.m., 2 p.m., and 7 117 p.m.), with either 30 mL of CDCM or placebo, both provided by Curaden AG (Kriens, 118

Switzerland) for 1 min (Appendix Figure 1 ). Participants were instructed to collect their saliva 119 by trained nurses using the "Saliva Collection System" kit (Greiner Bio-one, Kremsmünster, 120 Austria). Saliva sampling was performed on the first day at T1 (9 a.m., before the first 121 mouthwash) and then at T2 (1 p.m.) and T3 (6 p.m.). On the following 6 days, only one 122 sample was taken at 3 p.m. The rationale for performing the pure saliva sampling before and 123 not after the mouthwash was to collect the amount of viral load that has accumulated in the 124 previous hours following the previous rinsing. on a minimal viral load difference of 1 log 10 copies/mL between placebo and CDCM groups, 139 a common standard deviation of 2 log 10 copies/mL, a power of 0.9 and a type I error of 5%. It 140 was calculated at least 70 subjects per group. With an estimated drop-out rate of 25%, 88 141 subjects per group were required (unilateral test). 142

Intention to treat (ITT) analyses were performed on the imputed data from all randomized 143 patients using Multiple Imputation by Chained Equations, based on a Monte-Carlo Markov 144

Chain algorithm under missing at random data hypothesis. We have performed a paired 145 nonparametric Wilcoxon signed rank test with Bonferroni correction comparing the decrease 146 of viral load over time: T1 vs T2, T1 vs T3 and T1 vs day 7 in both groups. Then the two 147 groups were compared at each time thanks to a non-parametric Mann-Whitney U test. Finally, 148 a mixed effect linear model (viral load repeated data along time from day 1 T1 to day 7) was 149 performed with group (CDCM/placebo) as fixed effect and individuals as random effect. 150

Additionally, per-protocol (PP) analysis using the same methodology than ITT analyses 151 was performed based on participants with a complete set of outcome data at day 1. 152 A post-hoc subgroup analyses were performed on the datasets with Day 1 T1 values 153 starting at the first quartile (Q1, patients' baseline viral load higher than 2.94 log 10 copies/mL 154 (ITT) or 2.95 log10 copies/mL (PP)), the second quartile (Q2, patients' baseline viral load 155 higher than 4.01 log 10 copies/mL; 4.12 log10 copies/mL (PP)) and the third quartile (Q3, 156 patients' baseline viral load higher than 5.03 log 10 copies/mL; >5.16 log10 copies/mL (PP)). the symptoms on the COVID-19 report forms. Participants were randomized within 4 days 172 (IQR 3-5 days) of symptom onset. The first saliva specimens were collected at a median time 173 of 4 days (IQR 3-5 days) after nasopharyngeal PCR-positive results. Median initial viral load 174 was 4.01 log 10 copies/mL (IQR 2.94-5.03 log 10 copies/mL, range 0-10.19 log 10 copies/mL). 175

The first quartile (Q1) corresponded to a viral load starting at 2.94 log 10 copies/mL, whereas 176 the second (Q2) corresponded to a viral load starting at 4.01 log 10 copies/mL, and, the third 177 (Q3) corresponded to a viral load starting at 5.03 log 10 copies/mL. 178 179 Change in SARS-CoV-2 salivary viral load during the first day for all the patients 180

The SARS-CoV-2 salivary load continuously decreased between T1-T2 and T2-T3 for the 181 CDCM group and the placebo group ( Figure 2 ). The median viral load was lower in the 182 CDCM group compared to the placebo group at T2 and T3. A significant difference was 183 observed in viral load reduction in the before-after comparison of the same patients receiving 184 CDCM versus no difference for the placebo group from T1 to T2 (p=0.036) ( Table 2) For patients with an initially salivary SARS-CoV-2 load higher than 4.01 log 10 203 copies/mL, the results did not show a significant difference between CDCM and placebo at 204 T2 (p=0.182) or T3 (p=0.257). The median percentage decrease between T1 and T3 was -205 16.35% for CDCM (95% CI; -37.42% --3.56%) and -12.30% for placebo (95% CI; -27.19% -206 1.53%). 207

For patients with an initial SARS-CoV-2 saliva load higher than 5.03 log 10 copies/mL, 208 the quantitative results showed no significant difference between the 2 groups for periods T1-209 T2 and T1-T3. The median percentage decrease at T1-T3 was -24.14% for CDCM (95% CI; -210 41.05% --4.49%) and -14.15% for placebo (95% CI; -27.71% --4.21%). Similar results were 211 obtained with the per-protocol analysis (Appendix Table 6 ). The changes from baseline in the amount of SARS-CoV-2 in salivary samples at 7 days were 215 analyzed in intention-to-treat. 216 A continuous decrease over the 7 days for the CDCM group and the placebo group is 217 observed (Figure 3) . At day 7, no significant difference between patients receiving CDCM 218 and those receiving placebo was observed (p=0.388) ( Table 2 and Appendix Table 4 ). In both 219 groups, the viral load was significantly lower on day 7 than on day 1 T1 (p< 0.001). With the 220 linear mixed model, a higher but nonsignificant (p=0.112) reduction in the viral load was 221 observed in the CDCM group (mean difference -0.17 [90%CI -0.39 -0.06]). Similar results 222

were obtained with the per-protocol analysis (Appendix Table 4 , Figures 3 and 4) . 223 224

load 226 At day 7, the median salivary viral load was always lower for the CDCM group than for the 227 placebo group (Figure 3) . No significant differences between the two groups were observed 228 (Table 2) . For initial salivary loads greater than 5.03 log 10 copies/mL, the median percentage 229 decrease at day 7 compared with that at T1 for the CDCM group vs. the placebo group was - Table 5 , Figures 3 and 4) . The post-hoc analysis based on patients' baseline viral load led to several observations. 244

First, among participants with an initial load higher than 2.94 log 10 copies/mL, a significant 245 decrease in the CDCM group was observed in the 4 hours separating T2 from T1. Second, 246 their salivary viral load significantly decreased over the T1-T3 period in both groups; 247 however, there was a more positive impact in the CDCM group than in the placebo group. 248

The decrease observed in the placebo group could be explained by the natural decrease in 249 salivary viral load over the course of a day, by the effect of oral rinsing or by the presence of 250 excipients that can be considered as potential active ingredients [23] . Third, for patients with 251 initial loads greater than 4.01 log 10 copies/mL, a higher but nonsignificant percentage of viral 252 load reduction was observed for the CDCM group than for the placebo group. A hypothesis, 253 is that, for participants with the highest viral load, the frequency of mouthwash use could be 254 insufficient to significantly impact the viral load within a short period of time. 255

Moreover, the post-hoc analysis based on patient age concluded that the age was not 256 related to initial viral load at day 1 T1 (Spearman correlation test, p=0.07), nor to the 257 evolution up to day 1 T1 (Spearman correlation test, p=0.74). No impact over time was 258 observed (mixed linear model p=0.302). 259

Concerning the antiviral load responses to mouthwashes on day 7, our trial provided, 260 unclear evidence for the general population. By eliminating the fluctuation effect, there was a 261 greater drop in salivary viral load over time in the CDCM group. For patients with an initial 262 viral SARS-CoV-2 load higher than 4.01 log 10 copies/mL or than 5.03 log 10 copies/mL, 263 CDCM significantly reduced the salivary viral load more quickly than placebo. 264

Our study had limitations related to the time elapsed from the first salivary collection to 265 the time delay estimate for adults without clinical symptoms. 30% of individuals with 266 infection never develop symptoms [24, 25] . Since infectivity appears to peak at or before 267 symptom onset, the initial viral load data underestimate the salivary concentration load of the 268 general population during the incubation period [26] . Second, in our RCT, the proportion of 269 missing data was 12% (13% in the Active group; 11% in the Control group). The reasons for 270 missing data-completely random missing data, random missing data, and nonrandom missing 271 data were not specified. Third, in both groups, some participants had no SARS-CoV-2 load at 272 day 1 T1 (28/176, 15 Evolution for patients with a SARS-CoV-2 viral load greater than 2.94 log 10 copies/mL at day 1 T1. (D) SARS-CoV-2 viral load difference between T1 and T2 for patients with a SARS-CoV-2 viral load greater than 2.94 log 10 copies/mL at day 1 T1. (E) Evolution for patients with a SARS-CoV-2 viral load greater than 4.01 log 10 copies/mL at day 1 T1. (F) SARS-CoV-2 viral load difference between T1 and T2 for patients with a SARS-CoV-2 viral load greater than 4.01 log 10 copies/mL at day 1 T1. (G) Evolution for patients with a SARS-CoV-2 viral load greater than 5.03 log 10 copies/mL at day 1 T1. (H) SARS-CoV-2 viral load difference between T1 and T2 for patients with a SARS-CoV-2 viral load greater than 5.03 log 10 copies/mL at day 1 T1. Evolution of SARS-CoV-2 viral load for patients with a viral load greater than 2.94 log 10 copies/mL at day 1. (D) SARS-CoV-2 salivary viral load difference between day 1 time 1 and day 1 time 2 for patients with a viral load greater than 2.94 log 10 copies/mL at day 1 time 1. (E) Evolution of SARS-CoV-2 viral load for patients with a viral load greater than 4.01 log 10 copies/mL at day 1 time 1. (F) SARS-CoV-2 salivary viral load difference between day 1 time 1 and day 1 time 2 for patients with a viral load greater than 4.01 log 10 copies/mL at day 1 time 1. (G) Evolution for patients for patients with a viral load greater than 5.03 log 10 copies/mL at day 1 time 1. (H) SARS-CoV-2 salivary viral load difference between day 1 time 1 and day 1 time 2 for patients with a viral load greater than 5.03 log 10 copies/mL at day 1 time 1. Figure 3 . Evolution of SARS-CoV-2 salivary load within the mouthwash cohorts from day 1 to day 7 (intention-to-treat analysis) (A) Evolution for all the patients. (B) Evolution for patients for patients with a viral load greater than 2.94 log 10 copies/mL at day 1 time 1. (C) Evolution for patients for patients with a viral load greater than 4.01 log 10 copies/mL at day 1 time 1. (D) Evolution for patients for patients with a viral load greater than 5.03 log 10 copies/mL at day 1 time 1. 

specimen for testing respiratory virus by a point-of-care molecular assay: a 337 diagnostic validity study

Consistent detection of 2019 novel coronavirus in saliva

Clinical Significance 343 of a High SARS-CoV-2 Viral Load in the Saliva

Bioaerosol Spread of SARS-CoV-2 for the COVID-19 Pandemic

Transmission of COVID-19 349 virus by droplets and aerosols: A critical review on the unresolved dichotomy

Persistence of coronaviruses on 352 inanimate surfaces and their inactivation with biocidal agents

COVID-355 19: A Recommendation to Examine the Effect of Mouthrinses with β-Cyclodextrin Combined 356 with Citrox in Preventing Infection and Progression

Antiviral 359 Activity of Reagents in Mouth Rinses against SARS-CoV-2

Potential role of oral rinses targeting the viral lipid envelope in SARS-CoV-2 infection

Lowering the 365 transmission and spread of human coronavirus

Is the oral cavity relevant in SARS-CoV-2 368 pandemic?

Flavonoids as Antiviral Agents for Enterovirus A71 (EV-A71)

Structure-activity relationship of 372 flavonoid bifunctional inhibitors against Zika virus infection

Cyclodextrins: Emerging Medicines of the New Millennium

Flavonoid baicalin 377 inhibits HIV-1 infection at the level of viral entry

A Novel Sulfonated 383 Derivative of β-Cyclodextrin Effectively Inhibits Influenza A Virus Infection in vitro and in 384 vivo

Cyclodextrins in Antiviral 386 Therapeutics and Vaccines

Nasal Detection of the SARS-CoV-2 Virus After Antiviral Mouthrinses (BBCovid): A 390 structured summary of a study protocol for a randomised controlled trial

Simple classification of COVID-19 patients

The activities 395 of drug inactive ingredients on biological targets

SARS-CoV-2 Transmission From People Without COVID-19 Symptoms

Underdetection of cases of COVID-19 in France threatens epidemic control

Temporal dynamics in viral 404 shedding and transmissibility of COVID-19

All authors have completed the ICMJE uniform disclosure form. Denis Bourgeois reports 301 non-financial support and other from Curaden AG Switzerland, outside the submitted work. 302All other authors declare no competing interests. 303 304 Funding 305This work was partially supported by Curaden AG, Kriens, Switzerland and by Laboratory 306 "Systemic Health Care", EA4129, University of Lyon, France. The funder of the study had no 307 role in the study design, data collection, data analysis, data interpretation, or writing of the 308 report. All the authors have full access to all the data in the study and had final responsibility 309 for the decision to submit for publication. Luc Hospital (Lyon, France). We also thank all the technician from the CNR for their work. 319We thank Herve Morisset independent statistician and special adviser. 320We also extend our thanks to Eric Bomel, EZUS, University Lyon1 who provided central 321 administrative support to the project, Ursula Sutter from Greiner Bio-One GmbH (St. Gallen, 322Switzerland) and Dr. Eric Gonzalez Garcia, from Greiner Bio-One GmbH (