Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2

Abstract

Most public health measures to contain the COVID-19 pandemic are based on preventing the pathogen spread, and the use of oral antiseptics has been proposed as a strategy to reduce transmission risk. The aim of this manuscript is to test the efficacy of mouthwashes to reduce salivary viral load in vivo. This is a multi-centre, blinded, parallel-group, placebo-controlled randomised clinical trial that tests the effect of four mouthwashes (cetylpyridinium chloride, chlorhexidine, povidone-iodine and hydrogen peroxide) in SARS-CoV-2 salivary load measured by qPCR at baseline and 30, 60 and 120 min after the mouthrinse. A fifth group of patients used distilled water mouthrinse as a control. Eighty-four participants were recruited and divided into 12-15 per group. There were no statistically significant changes in salivary viral load after the use of the different mouthwashes. Although oral antiseptics have shown virucidal effects in vitro, our data show that salivary viral load in COVID-19 patients was not affected by the tested treatments. This could reflect that those mouthwashes are not effective in vivo, or that viral particles are not infective but viral RNA is still detected by PCR. Viral infectivity studies after the use of mouthwashes are therefore required. ( https://clinicaltrials.gov/ct2/show/NCT04707742 ; Identifier: NCT04707742).

Figure 1

Diagram of the experimental study protocol. The upper left panel illustrates the multicentre study with five hospitals involved, the number of enrolled patients diagnosed and hospitalized because of SARS-COV-2 infection and the record of clinical variables. The sampling of the four non stimulated saliva samples for each patient is represented by the numbers 1, 2, 3 and 4 (corresponding to one basal and three post-mouthwash samples, at 30 min, at 60 min and at 120 min, respectively), as well as the rinsing time for 1 min. In this blind study, participants were randomly assigned to one of the five parallel group: PVP-I (group A), H₂O₂ (group B), CPC (group C), CHX (group D) and distilled water (group E, control group). After each sampling, 0.5 mL of saliva was transferred to a sterile Eppendorf tube with 1.5 mL of virus inactivating buffer, illustrated in the figure with a syringe. All samples were taken to the laboratory where RNA extraction and RT-qPCR (reverse transcription—quantitative polymerase chain reaction) was performed.

Figure 2

Trial profile. Boxes display the number of individuals recruited for the clinical study and those assigned to each treatment, indicating the number of samples discarded due to undetectable viral load. ^aNo viral load detected at all four times. ^bNo viral load detected in any one of the four sampling times. *Unpaired statistical analysis (three samples per patient available, including basal values). **Paired statistical analysis (all four samples available).

Figure 3

Salivary viral loads for all five treatment groups through time. Box plots show viral load levels, as measured by qPCR, for each of the patients in the group A—PVP-I (povidone-iodine), B—Hydrogen peroxide (H₂O₂), C—CPC (cetylpyridinium chloride), D—CHX (chlorhexidine) and E—Distilled water (placebo group) at the four different timepoints (1 for basal and 2, 3 and 4 for the three times after the mouthwash, corresponding to 30, 60 and 120 min after the mouthwash, respectively). Lines joining the samples from the same patient are also shown.

Figure 4

Relative changes in salivary viral load for all five treatment groups after the mouthwash. Box plots show changes of viral load (%) for each of the patients in the group A—PVP-I (povidone-iodine), B—H₂O₂, C—CPC (cetylpyridinium chloride), D—CHX (chlorhexidine) and E (placebo group) at the three different times after the oral rinse treatment, relative to the baseline viral load prior to the mouthwash (changes at 30 min: t2–t1; changes at 60 min: t3–t1; changes at 120 min: t4–t1). Lines joining the points from the same patient through time are also shown.

Figure 5

Patients with improvement in viral load values. Bar plots show the percentage of individuals lowering salivary viral load by 50% or higher (a), or 90% or higher (b) relative to baseline values prior to the oral rinsing treatment, at different times after the mouthwash (t2: 30 min, t3: 60 min and t4: 120 min) in each study group: PVP-I (povidone-iodine), H₂O₂, CPC (cetylpyridinium chloride), CHX (chlorhexidine) and control group, respectively.