Effect of oral antiseptics in reducing SARS-CoV-2 infectivity: evidence from a randomized double-blind clinical trial

Abstract

ABSTRACTBackground: In vitro studies have shown that several oral antiseptics have virucidal activity against SARS-CoV-2. Thus, mouthwashes have been proposed as an easy to implement strategy to reduce viral transmission. However, there are no data measuring SARS-CoV-2 viability after mouthwashes in vivo.

Methods: In this randomized double-blind, five-parallel-group, placebo-controlled clinical trial, SARS-CoV-2 salivary viral load (by quantitative PCR) and its infectious capacity (incubating saliva in cell cultures) have been evaluated before and after four different antiseptic mouthwashes and placebo in 54 COVID-19 patients.

Results: Contrary to in vitro evidence, salivary viral load was not affected by any of the four tested mouthwashes. Viral culture indicated that cetylpyridinium chloride (CPC) significantly reduced viral infectivity, but only at 1-hour post-mouthwash.

Conclusion: These results indicate that some of the mouthwashes currently used to reduce viral infectivity are not efficient in vivo and, furthermore, that this effect is not immediate, generating a false sense of security.Trial registration: ClinicalTrials.gov identifier: NCT04707742..

Keywords: COVID-19; SARS-CoV-2; infectivity; mouthwash; saliva.

Figure 1.

Trial profile. Diagram showing the number of patients enrolled and randomized, excluded for not having sufficient saliva volume or negative to RT-qPCR in the baseline samples, and the total evaluated patients in which total viral load (orange square) and viable (infective) viral load (blue square) were determined. The numbers of inpatients assigned to each of the different treatment groups are represented inside boxes of different colours. PVP-I (povidone-iodine) in blue, Hydrogen peroxide (H₂O₂) in red, CPC (cetylpyridinium chloride) in purple, CHX (chlorhexidine) in orange, and Placebo (distilled water) in green. t1: basal time point. ^a Number of patients excluded from the cell culture assays for low salivary viral load (Ct value > 35 in the RT-qPCRs in the baseline saliva sample). ^b Number of patients without detectable viral load after cell culture in the baseline saliva sample.

Figure 2.

Positive viral culture as a function of salivary viral load. Bars represent the percentage of SARS-CoV-2 positive viral culture of in saliva samples from COVID-19 inpatients. Saliva samples were incubated in Vero-E6 cells for 1 h to allow viral adsorption and then replaced by fresh culture medium. At day 5 post-infection, viral replication was measured by CPE reading and SARS-CoV-2 RNA quantitation in the culture supernatant. A culture was considered positive by RT-qPCR Ct values < 37 in day 5 post-infection supernatant (equivalent to ≥ 2 × 10³ SARS-CoV-2 copies per mL). The number of patients in each Ct range was: n = 3 in Ct range 15-19, n = 10 in Ct ranges 20–24 and 29–31, n = 9 in Ct range 25–28 and n = 8 in Ct range 32–34.

Figure 3.

Salivary viral loads. Box plots represent the median values of viral loads in log copies per mL of saliva measured by RT-qPCR for basal saliva (t1), 30 min (t2) and one hour (t3) after the oral rinse for each treatment group: PVP-I (povidone-iodine), Hydrogen peroxide (H₂O₂), CPC (cetylpyridinium chloride), CHX (chlorhexidine) and PLACEBO (distilled water). The dotted lines join the values for the same patient through time. Different y-axis scales are used, for clarity. * Wilcoxon paired test (p value = .048).

Figure 4.

SARS-CoV-2 genome copies/mL in supernatants from day 5 virus culture in Vero-E6 cells (median values in log copies per mL of culture supernatant measured by RT-qPCR). Box plots are represented for each treatment group (PVP-I (povidone-iodine), Hydrogen peroxide (H₂O₂), CPC (cetylpyridinium chloride), CHX (chlorhexidine) and PLACEBO (distilled water)) at each time point (t1 for basal and t2 and t3 for 30 and 60 min after the mouthwash, respectively). The values for each patient are linked over time by dotted lines. Different y-axis scales are used, for clarity. * Wilcoxon paired test (p value = .015).

Figure 5.

Outline of the clinical trial protocol. From top to bottom, the upper left box represents: the number of hospitals involved in this multicenter study, the number of enrolled and randomized inpatients and the registry of their clinical variables. On the right, the timeline protocol for collecting the three non-stimulated saliva samples is illustrated, before (1) and 30 min and one hour after (2 and 3, respectively) the one-minute oral rinse with the respective mouthwash. All samples were taken to the laboratory where each saliva sample was divided into two aliquots, one to determine the viral load per mL of saliva, by means of its RNA extraction and subsequent RT-qPCR (reverse transcription-quantitative polymerase chain reaction), and the other to assess viral infectivity in Vero-E6 cell culture, by detection of cytopathic effect (CPE) and viral replication (RNA extraction and RT-qPCR of the virus culture supernatant).