Contemporary Update on Pre-procedural Rinsing

Contemporary Update on Pre-procedural Rinsing

Antimicrobial rinses have historically been used off-label pre-procedurally as an infection control adjunct during aerosol-generating procedures.1Marui VC, Souto MLS, Rovai ES et al. Efficacy of preprocedural mouthrinses in the reduction of microorganisms in aerosol: A systematic review. J Am Dent Assoc 2019;150(12):1015-26.e1. doi: 10.1016/j.adaj.2019.06.024.^,2Sreenivasan PK, Gittins E. The effects of a chlorhexidine mouthrinse on culturable micro-organisms of the tongue and saliva. Microbiol Res 2004;159(4):365-70.^,3Larsen PE. The effect of a chlorhexidine rinse on the incidence of alveolar osteitis following the surgical removal of impacted mandibular third molars. J Oral Maxillofac Surg 1991;49(9):932-37.^,4Hennessy B, Joyce A. A survey of preprocedural antiseptic mouth rinse use in Army dental clinics. Mil Med 2004;169(8):600-3. More recently, pre-procedural rinsing recommendations and their implementation were propelled during the COVID-19 pandemic, in this case with the objective of reducing viral loads of SARS-CoV-2.5American Dental Association. Interim Guidance for Minimizing Risk of COVID-19 Transmission. https://www.ada.org/~/media/CPS/Files/COVID/ADA_COVID_Int_Guidance_Treat_Pts.pdf. ^,6Meng L, Fang H, Bian Z. Coronavirus Disease 2019 (COVID-19): Emerging and Future Challenges for Dental and Oral Medicine. J Dent Res 2020;99. 002203452091424. 10.1177/0022034520914246.^,7Peng X, Xu X, Li Y et al. Transmission routes of 2019-nCoV and controls in dental practice. Int J Oral Sci 2020;12:9. Chlorhexidine gluconate (CHX), cetylpyridinium chloride (CPC), essential oils (EO), povidone-iodine (PVP-I) and hydrogen peroxide (HP) are the main mouthrinses used in this manner.1Marui VC, Souto MLS, Rovai ES et al. Efficacy of preprocedural mouthrinses in the reduction of microorganisms in aerosol: A systematic review. J Am Dent Assoc 2019;150(12):1015-26.e1. doi: 10.1016/j.adaj.2019.06.024.^,8Feres M, Figueiredo LC, Faveri M et al. The effectiveness of a preprocedural mouthrinse containing cetylpyridinium chloride in reducing bacteria in the dental office. J Am Dent Assoc 2010;141:415-22.^,9Domingo MA, Farrales MS, Loya RM et al. The effect of 1% povidone-iodine as a pre-procedural mouthrinse in 20 patients with varying degrees of oral hygiene. J Philipp Dent Assoc 1996;48(2):31-8. In this article, we will briefly look at the mechanisms of action for rinses and research on their antibacterial efficacy, then focus on findings on the antiviral efficacy of antimicrobial rinses.

Bactericidal and virucidal mechanisms of action
At bactericidal concentrations, CHX and CPC disrupt cell membranes, resulting in leakage of intracellular proteins and cell death, while EO rinses are generally believed to also disrupt bacterial cell membranes and to inhibit bacterial enzymatic activity.10DePaola LG, Eshenaur Spolarich A. Safety and Efficacy of Antimicrobial Mouthrinses in Clinical Practice. J Dent Hyg 2007;81(suppl 1) 117. ^,11Scheie AA. Modes of action of currently known chemical antiplaque agents other than chlorhexidine. J Dent Res 1989;68 (Spec Iss):1609-16.^,12Nazzaro F, Fratianni F, De Martino L et al. Effect of essential oils on pathogenic bacteria. Pharmaceuticals (Basel) 2013;6(12):1451‐74. doi:10.3390/ph6121451.^,13Weber J, Bonn EL, Auer DL et al. Preprocedural mouthwashes for infection control in dentistry-an update. Clin Oral Investig 2023;27(Suppl 1):33-44. doi: 10.1007/s00784-023-04953-z. Oxidizing agents include PVP-I, which releases free (unbound) iodine that penetrates cell membranes and interacts with proteins and fatty acids, and HP which releases hydroxyl radicals – again resulting in cell death.14Litsky BY, Mascis JD, Litsky W. Use of an antimicrobial mouthwash to minimize the bacterial aerosol contamination generated by the high-speed drill. PlumX Metrics. https://doi.org/10.1016/0030-4220(70)90407-X.

The virucidal effect of CPC, PVP-I, benzalkonium chloride (BAC), and CHX on enveloped viruses consists of disruption of the viral envelope, a lipid layer essential for viral cell functioning.15Cieplik F, Jakubovics NS. Preprocedural Mouthwashes for Reduction of SARS-CoV-2 Viral Load and Infectivity. J Dent Res 2022;101(12):1421-3. doi:10.1177/00220345221110444.^,16Muñoz-Basagoiti J, Perez-Zsolt D, León R et al. Mouthwashes with CPC Reduce the Infectivity of SARS-CoV-2 Variants In Vitro. J Dent Res 2021;100(11):1265-72. doi: 10.1177/00220345211029269.^,17Popkin DL, Zilka S, Dimaano M et al. Cetylpyridinium Chloride (CPC) Exhibits Potent, Rapid Activity Against Influenza Viruses in vitro and in vivo. Pathog Immun 2017;2(2):252-9. doi: 10.20411/pai.v2i2.200. The same mechanism of action is believed to occur with EO rinses, based on in vitro tests showing EO efficacy against enveloped viruses.18Dennison D K, Meredith G M, Shillitoe E J, Caffesse R G. The antiviral spectrum of Listerine antiseptic. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:442-8. Disruption of the viral envelope by PVP-I and HP is the result of oxidation, and both also attack viral genetic material.7Peng X, Xu X, Li Y et al. Transmission routes of 2019-nCoV and controls in dental practice. Int J Oral Sci 2020;12:9.^,19Kirk-Bailey J, Combes J, Sunkaraneni S, Challacombe S. The use of Povidone Iodine nasal spray and mouthwash during the current COVID-19 pandemic for the reduction of cross infection and protection of healthcare workers. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3563092.^,20Centers for Disease Control and Prevention. Chemical Disinfectants Guideline for Disinfection and Sterilization in Healthcare Facilities (2008). https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-methods/chemical.html#Hydrogen.^,21Eggers M. Infectious Disease Management and Control with Povidone Iodine. Infect Dis Ther 2019;8:581-93.^,22Kanagalingam J, Feliciano R, Hah JH et al. Practical use of povidone-iodine antiseptic in the maintenance of oral health and in the prevention and treatment of common oropharyngeal infections. Int J Clin Pract 2015;69(11):1247-56. doi: 10.1111/ijcp.12707. (Figures 1a,b)

Figure 1a. Bactericidal activity of antimicrobial rinses

Figure 1b: Effect on enveloped viruses: Oxidizing agents, CHX, CPC, other

Bactericidal efficacy of pre-procedural rinsing

Several systematic reviews have confirmed reductions in bacterial loads after using antimicrobial mouthrinses. In a systematic review of 13 randomized controlled trials (RCT) evaluating the efficacy of pre-procedural rinsing in reducing bacterial loads in dental procedure-generated aerosols compared to controls (water, placebo or no rinse), the number of colony-forming units per milliliter (CFU/ml) was reduced by a mean of 78.9%, 61.3% and 61.2%, respectively, for CHX, EO and CPC rinses.1Marui VC, Souto MLS, Rovai ES et al. Efficacy of preprocedural mouthrinses in the reduction of microorganisms in aerosol: A systematic review. J Am Dent Assoc 2019;150(12):1015-26.e1. doi: 10.1016/j.adaj.2019.06.024. In a second systematic review with twenty RCT, 15 studies assessed the effect of pre-procedural rinsing with CHX for between 30 seconds and 2 minutes.23Mohd-Said S, Mohd-Dom TN, Suhaimi N et al. Effectiveness of Pre-procedural Mouth Rinses in Reducing Aerosol Contamination During Periodontal Prophylaxis: A Systematic Review. Front Med 2021;8:600769. doi: 10.3389/fmed.2021.600769. In over half of these, a more than 70% reduction in CFU/ml was observed for the test group vs the control group. Thirdly, in a review published in the Spring of 2023 the researchers determined that pre-procedural mouthrinses containing CHX, CPC or EO can significantly reduce bacterial loads in procedure-generated aerosols.13Weber J, Bonn EL, Auer DL et al. Preprocedural mouthwashes for infection control in dentistry-an update. Clin Oral Investig 2023;27(Suppl 1):33-44. doi: 10.1007/s00784-023-04953-z.

Anti-viral efficacy in enveloped and non-enveloped viruses

Research on the anti-viral efficacy of mouthrinse was limited prior to the COVID-19 pandemic. In an in vitro study conducted in 1990, 30-second applications of 0.12% CHX to tissue cultures resulted in a 97% reduction in the herpes simplex virus (HSV-1), an enveloped virus.24Bernstein D, Schiff G, Echler G et al. In vitro Virucidal Effectiveness of a 0.12%-Chlorhexidine Gluconate Mouthrinse. J Dent Res 1990;69(3):874-876. doi:10.1177/00220345900690030901. Additionally, efficacy was observed against other enveloped viruses, including influenza A, parainfluenza, cytomegalovirus and hepatitis B virus, while CHX was ineffective against polio virus (a non-enveloped virus). Other in vitro studies support these findings, including a study in which flow cytometry and cell quantification showed that applications of 0.05% CPC plus 0.12% CHX or 0.07% CPC to tissue cultures resulted in anti-viral effects against HSV but not HPV (a non-enveloped virus).25Riveira-Muñoz E, Garcia-Vidal E, Bañó-Polo M et al. Cetylpyridinium Chloride-Containing Mouthwashes Show Virucidal Activity against Herpes Simplex Virus Type 1. Viruses 2023;15:1433. https://doi.org/10.3390/v15071433. In a third study, 0.12% CHX and EO rinses inhibited HSV-1 and HIV-1 following application for at least 30 seconds, and influenza virus was inactivated by low-concentration CPC.17Popkin DL, Zilka S, Dimaano M et al. Cetylpyridinium Chloride (CPC) Exhibits Potent, Rapid Activity Against Influenza Viruses in vitro and in vivo. Pathog Immun 2017;2(2):252-9. doi: 10.20411/pai.v2i2.200. Similarly, in a fourth study (1997), CHX, alkyldiamino-ethyl-glycine hydrochloride, BAC and benzethonium chloride offered anti-viral efficacy against HIV, rubella, measles and mumps viruses, while not against non-enveloped viruses (adenovirus, polio virus and rhinoviruses).26Kawana R, Kitamura T, Nakagomi O et al. Inactivation of Human Viruses by Povidone-Iodine in Comparison with Other Antiseptics. Dermatol 1997;195 (Suppl. 2):29-5. https://doi.org/10.1159/000246027 However, PVP-I was found to inactivate enveloped and non-enveloped viruses. In a RCT conducted in 2005 in two phases, after patients with active herpes labialis lesions rinsed for 30 seconds with an EO mouthrinse, the viral titer of HSV in salivary samples was eliminated shortly after and 30 minutes post-rinsing.27Meiller TF, Silva A, Ferreira SM et al. Efficacy of Listerine® Antiseptic in reducing viral contamination of saliva. J Clin Periodontol 2005;32:341-6. https://doi.org/10.1111/j.1600-051X.2005.00673.x. In contrast, a sample taken 60 minutes post-rinsing in the second phase of the study showed a non-significant difference in the viral titer compared to baseline.

Test methods for viral detection and anti-viral efficacy
Traditionally, in vitro studies have used plaque assays, or determined the tissue culture infectious dose fifty (TCID50), to measure viral titers for evaluation of the effect of anti-viral agents on viral infectivity, including mouthrinses.28Welch SR, Davies KA, Buczkowski H et al. Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives. J Clin Microbiol 2020;58(11):e01713-20. doi: 10.1128/JCM.01713-20.^,29Hong W, Xiong J, Nyaruaba R et al. Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. Sci Total Environ 2021;797:149085. doi: 10.1016/j.scitotenv.2021.149085.^,30Fernandez MDS, Guedes MIF, Langa GPJ et al. Virucidal efficacy of chlorhexidine: a systematic review. Odontol 2022;110(2):376-92. doi: 10.1007/s10266-021-00660-x. In the plaque assay test, cells are infected and then incubated, during which visible plaques form and are then counted, while TCID50 represents the dilution that results in alterations to viral structure in 50 percent of the cell cultures.31Moretti M. Viral Titer Quantification: Balancing Traditional and Modern Approaches for Comprehensive Analysis. Dec 26, 2023. https://www.labmanager.com/viral-titer-quantification-balancing-traditional-and-modern-approaches-for-comprehensive-analysis-31559. These tests are regularly used in vitro, while difficult to perform, time-consuming and costly.29Hong W, Xiong J, Nyaruaba R et al. Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. Sci Total Environ 2021;797:149085. doi: 10.1016/j.scitotenv.2021.149085.^,32Bonn EL, Rohrhofer A, Audebert FX et al. Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2–Positive Patients: A Randomized Controlled Clinical Trial. J Dent Res 2023;102(6):608-15. doi:10.1177/00220345231156415.

In vitro tests performed on patient samples to confirm COVID-19 infection include, but are not limited to, viral RNA-mediated reverse transcription polymerase chain reaction (RT-PCR), and ELISA tests which first require lysis of the virus to enable detection of proteins such as the nucleocapsid protein (N-protein).29Hong W, Xiong J, Nyaruaba R et al. Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. Sci Total Environ 2021;797:149085. doi: 10.1016/j.scitotenv.2021.149085.^,31Moretti M. Viral Titer Quantification: Balancing Traditional and Modern Approaches for Comprehensive Analysis. Dec 26, 2023. https://www.labmanager.com/viral-titer-quantification-balancing-traditional-and-modern-approaches-for-comprehensive-analysis-31559.^,33Taylor A, Calvez R, Atkins M, Fink CG. Comparing lateral flow testing with a rapid RT-PCR method for SARS-CoV-2 detection in the United Kingdom-A retrospective diagnostic accuracy study. Health Sci Rep 2022;5(5):e811. doi: 10.1002/hsr2.811. When performing RT-PCR testing, viral RNA – which is unstable – is first converted to complementary DNA (cDNA) by reverse transcription,34What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR? https://www.enzo.com/note/what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr/. and the number of cycles in a RT-PCR assay needed to amplify the cDNA is then computed to obtain cycle threshold (CT) values. These CT values are used as a proxy for viral loads, with an inverse relationship, i.e., lower CT values indicate higher viral loads (viral infectivity).35Finks SW, Van Matre E, Budd W et al. Clinical Significance of Quantitative Viral Load in Patients Positive for SARS-CoV-2. Am J Med Open 2023;10:100050. doi: 10.1016/j.ajmo.2023.100050.^,36Michalakis Y, Sofonea MT, Alizon S, Bravo IG. SARS-CoV-2 viral RNA levels are not 'viral load'. Trends Microbiol 2021;29(11):970-2. doi: 10.1016/j.tim.2021.08.008. ^,37Park J, Cho SI, Kang SG et al. Long-term trends in cycle threshold values: a comprehensive analysis of COVID-19 dynamics, viral load, and reproduction number in South Korea. Front Public Health 2024;12:1394565. doi: 10.3389/fpubh.2024.1394565.^,38Bhavnani D, James ER, Johnson KE et al. SARS-CoV-2 viral load is associated with risk of transmission to household and community contacts. BMC Infect Dis 2022;22(1):672. doi: 10.1186/s12879-022-07663-1.^,39Lee LYW, Rozmanowski S, Pang M et al. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infectivity by Viral Load, S Gene Variants and Demographic Factors, and the Utility of Lateral Flow Devices to Prevent Transmission. Clin Infect Dis 2022;74(3):407-15. doi: 10.1093/cid/ciab421.^,40Harris T, Geard N, Zachreson C. Correlation of viral loads in disease transmission could affect early estimates of the reproduction number. J R Soc Interface 2023;20(202):20220827. doi: 10.1098/rsif.2022.0827.

Figure 2. In vitro tests to assess viral loads

Low CT values are significantly related to the number of new cases among contacts of an infected individual. Quantitative RT-PCR (RT-qPCR), also referred to as real-time RT-PCR, is faster than RT-PCR, provides real-time results as the test progresses, and quantifies nucleic acids.34What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR? https://www.enzo.com/note/what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr/.

For in vivo studies, typically, RT-PCR and RT-qPCR testing has been performed rather than the more difficult and costly effort involved in rescuing virus using cell cultures.32Bonn EL, Rohrhofer A, Audebert FX et al. Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2–Positive Patients: A Randomized Controlled Clinical Trial. J Dent Res 2023;102(6):608-15. doi:10.1177/00220345231156415. Mechanistically, RT-PCR does evaluate viral RNA particles, however several researchers have noted that it does not determine whether the virus is viable or non-viable (dead).15Cieplik F, Jakubovics NS. Preprocedural Mouthwashes for Reduction of SARS-CoV-2 Viral Load and Infectivity. J Dent Res 2022;101(12):1421-3. doi:10.1177/00220345221110444.^,29Hong W, Xiong J, Nyaruaba R et al. Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. Sci Total Environ 2021;797:149085. doi: 10.1016/j.scitotenv.2021.149085.^,36Michalakis Y, Sofonea MT, Alizon S, Bravo IG. SARS-CoV-2 viral RNA levels are not 'viral load'. Trends Microbiol 2021;29(11):970-2. doi: 10.1016/j.tim.2021.08.008. As such, testing result differences between baseline and post-interventions may not be reflective of viral load reductions.32Bonn EL, Rohrhofer A, Audebert FX et al. Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2–Positive Patients: A Randomized Controlled Clinical Trial. J Dent Res 2023;102(6):608-15. doi:10.1177/00220345231156415.^,41Ferrer MD, Barrueco AS, Martinez-Beneyto Y et al. Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2. Sci Rep 2021;11(1):24392. https://doi.org/ 10.1038/s41598-021-03461-y.^,42Tarragó-Gil R, Gil-Mosteo MJ, Aza-Pascual-Salcedo M et al. Randomized clinical trial to assess the impact of oral intervention with cetylpyridinium chloride to reduce salivary SARS-CoV-2 viral load. J Clin Periodontol 2023;50(3):288-94. https://doi.org/10.1111/jcpe.13746

In several more recent in vivo studies, cell culture tests or new test methods have been used to evaluate reductions in viral infectivity. One method is a modified enzyme-linked immunosorbent assay (ELISA) test (i.e., without performing the lysis step), to detect and quantify nucleocapsid protein levels released after viral envelope destruction.41Ferrer MD, Barrueco AS, Martinez-Beneyto Y et al. Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2. Sci Rep 2021;11(1):24392. https://doi.org/ 10.1038/s41598-021-03461-y. (Figure 2)

In vitro research on mouthrinse efficacy against SARS-CoV-2

SARS-CoV-2 is an enveloped virus with a single RNA strand. Sixteen in vitro studies were included in one systematic review, and segregated from RCTs for analyses, leading to the researchers finding that CPC, EO and PVP-I rinses offer antiviral efficacy.43Mezarina Mendoza JPI, Trelles Ubillús BP, Salcedo Bolívar GT et al. Antiviral effect of mouthwashes against SARS-COV-2: A systematic review. Saudi Dent J 2022;34(3):167-93. doi: 10.1016/j.sdentj.2022.01.006. Among individual in vitro studies, in one (2021), the infectivity of SARS-CoV-2 was significantly reduced by the application of HP, PVP-I, CHX, and alcohol-containing EO mouthrinses, with the greatest effect observed for HP, followed by PVP-I.44Xu C, Wang A, Hoskin ER et al. Differential Effects of Antiseptic Mouth Rinses on SARS-CoV-2 Infectivity In Vitro. Pathogens 2021;10(3):272. https://doi.org/10.3390/pathogens10030272. In a prior in vitro study (2020), the infectivity of three strains of SARS-CoV-2 was reduced by mouthrinse formulations containing CHX, PVP-I, chlorine dioxide, BAC, HP, EO, octenidine dihydrochloride, or polyhexanide.45Meister TL, Brüggemann Y, Todt D et al. Virucidal Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome Coronavirus 2. J Infect Dis 2020;222(8):1289-92. https://doi.org/10.1093/infdis/jiaa471.

Systematic reviews on anti-viral efficacy of rinses against SARS-CoV-2

Several systematic reviews have been conducted to evaluate the anti-viral efficacy of mouthrinses. In a recent systematic review that included 9 studies (RCT, NRCT and quasi-experimental), rinsing with 1% PVP-I was found to be effective in reducing salivary viral loads of SARS-CoV-2 (determined by obtaining CT values), with no significant effect observed for CPC or CHX.46Ebrahimi T, Shamshiri AR, Alebouyeh M, Mohebbi SZ. Effectiveness of mouthwashes on reducing SARS-CoV-2 viral load in oral cavity: a systematic review and meta-analysis. BMC Oral Health 2023;23(1):443. In contrast, in a systematic review with 22 RCT and 1 NRCT published up to March 2023, only five RCT were eligible for a network meta-analysis. No statistically significant reductions in viral loads were found after rinsing with sodium chloride (NaCl), PVP-I, ß-cyclodextrin + citrox, HP, CHX, CPC, hypochlorous acid (HOCl) or placebo, based on RT-PCR tests.47Lin SY, Sun JS, Hung MC, Chang JZ. Effectiveness of mouth rinses against COVID-19: a systematic review and network meta-analysis. J Hosp Infect 2023;139:175-91. doi: 10.1016/j.jhin.2023.06.022. Among RCT and cohort studies in another systematic review, eight studies included CHX, eight included PVP-I and four included HP.48Ziaeefar P, Bostanghadiri N, Yousefzadeh P et al. The efficacy of mouthwashes in reducing SARS-CoV-2 viral loads in human saliva: A systematic review. New Microbes New Infect 2022;49:101064. doi: 10.1016/j.nmni.2022.101064. Among these, the outcomes were mixed: using RT-PCR, significant reductions in salivary viral loads were found in 5 studies each after rinsing with CHX or PVP-I, and in 2 of 4 studies investigating HP.

In a systematic review of 13 RCT published up to January, 2023, in seven RCT the CT values significantly increased (proxy for viral load reductions) after rinsing with CHX, PVP-I or HP.49Zhang M, Meng N, Duo H et al. Efficacy of mouthwash on reducing salivary SARS-CoV-2 viral load and clinical symptoms: a systematic review and meta-analysis. BMC Infect Dis 2023;23(1):678. doi: 10.1186/s12879-023-08669-z. Three studies in the review used RT-PCR plus additional testing using either cell cultures or testing for nucleocapsid protein levels. In all three studies, significant differences were found using these tests, but not RT-PCR tests. In a second systematic review of 6 RCT, it was found that CPC was effective in reducing salivary viral loads in vivo.50D'Amico F, Moro M, Saracino M et al. Efficacy of Cetylpyridinium Chloride mouthwash against SARS-CoV-2: A systematic review of randomized controlled trials. Mol Oral Microbiol 2023;38(3):171-80. doi: 10.1111/omi.12408.

Other systematic reviews have included both in vivo and in vitro studies. For example, in one with 25 studies, four were RCT and the remainder in vitro studies, and it was concluded that CHX had the ability to briefly reduce the intra-oral viral load.30Fernandez MDS, Guedes MIF, Langa GPJ et al. Virucidal efficacy of chlorhexidine: a systematic review. Odontol 2022;110(2):376-92. doi: 10.1007/s10266-021-00660-x. In another systematic review and meta-analysis, 14 RCT and 21 in vitro studies were included.51Sbricoli L, Schiavon L, Brunello G et al. Efficacy of different mouthwashes against COVID-19: A systematic review and network meta-analysis. Jpn Dent Sci Rev 2023;59:334-56. doi: 10.1016/j.jdsr.2023.09.003. It was concluded that the results supported the effectiveness of CHX, PVP-I, CPC and HP, while only CHX resulted in a significant difference in CT values 5 minutes post-rinsing compared to the control. However, a high risk of bias was found for a number of studies.51Sbricoli L, Schiavon L, Brunello G et al. Efficacy of different mouthwashes against COVID-19: A systematic review and network meta-analysis. Jpn Dent Sci Rev 2023;59:334-56. doi: 10.1016/j.jdsr.2023.09.003. In another systematic review, 16 in vitro studies and 11 clinical trials were included, and segregated for analyses.43Mezarina Mendoza JPI, Trelles Ubillús BP, Salcedo Bolívar GT et al. Antiviral effect of mouthwashes against SARS-COV-2: A systematic review. Saudi Dent J 2022;34(3):167-93. doi: 10.1016/j.sdentj.2022.01.006. For the in vivo studies HP, CHX, PVP-I, CPC, β-cyclodextrin-citrox, and sorbitol with xylitol were found to be effective. All in vivo studies used RT-PCR testing, while one additionally used TCID50. However, in three studies including this one, samples were obtained after multiple days which is not relevant for pre-procedural rinsing.

Lastly, in a review of 5 studies published up to February 2021, in four studies the use of PVP-I, CHX, or CPC mouthrinse was found to be able to reduce salivary viral loads.52Giulia B, Viktoria W, Robert K et al. Eligibility and efficacy of a CPC- and CHX-based antiviral mouthwash for the elimination of SARS-CoV-2 from the saliva: a randomized, double-blind, controlled clinical trial. J Clin Periodontol 2024;51(2):158-66. The researchers noted that they could not draw conclusions due to study heterogeneity and findings of a high risk of bias in several due to factors including confounders, patient selection, missing data, and selective reporting.

In vivo research on mouthrinse efficacy using RT-PCR testing

In a recent RCT, RT‐qPCR was used to analyze salivary samples from subjects at baseline prior to rinsing, as well as 5, 30 and 60 minutes after rinsing with 15 ml of 1% PVP-I, 1.5% HP, 0.075% CPC, or 80 ppm HOCl for 30 seconds.53Alzahrani MM, Bamashmous S, Alkharobi H et al. Mouth rinses efficacy on salivary SARS-CoV-2 viral load: A randomized clinical trial. J Med Virol 2023;95(1):e28412. doi: 10.1002/jmv.28412. CPC, PVP-I and HP all resulted in significant reductions in salivary viral loads 30 and 60 minutes post-rinsing, and for HP a reduction was also observed 5 minutes post-rinsing. In another RCT (2021) with 16 patients, similar salivary viral load reductions were found for CPC, PI and CHX based on CT values. Rinsing with CHX, CPC or PVP-I, or water (control), with saliva samples collected at baseline, and at 5 minutes, 3 and 6 hours in another study was found to result in similar salivary viral load reductions for CPC, PI and CHX mouthrinses, based on CT values. After computing CT value fold reductions, significantly greater reductions were found for CPC rinse at 5 minutes and 6 hours compared to placebo, and at 6 hours for the PVP-I rinse.54Seneviratne CJ, Balan P, Ko KKK et al. Efficacy of commercial mouth-rinses on SARS-CoV-2 viral load in saliva: randomized control trial in Singapore. Infection 2021;49:305-11. https://doi.org/10.1007/s15010-020-01563-9. In a third RCT, rinsing for 60 seconds with 1% HP, 0.12% CHX or 0.5% PVP-I resulted in decreases in viral loads at 15 and 45 minutes after rinsing.55Chaudhary P, Melkonyan A, Meethil A et al. Estimating salivary carriage of severe acute respiratory syndrome coronavirus 2 in nonsymptomatic people and efficacy of mouthrinse in reducing viral load: A randomized controlled trial. J Am Dent Assoc 2021;152(11):903-8. doi: 10.1016/j.adaj.2021.05.021.

In one pilot study, patients rinsed in accordance with the manufacturers’ instructions with 0.075% CPC plus 0.28% Zinc lactate (Zn), HP, CHD, or sequentially with HP then CHX, or water (placebo).56Eduardo FP, Corrêa L, Heller D, et al. Salivary SARS-CoV-2 load reduction with mouthwash use: A randomized pilot clinical trial. Heliyon 2021;7(6):e07346. Salivary samples were taken at baseline, immediately after rinsing, and at 30- and 60-minutes post-rinsing. Significant reductions were found for CPC+Zn and CHX rinses at all three time-points, while the greatest reductions in viral load immediately after rinsing were observed with CPC+Zn and HP. Rinsing sequentially with HP and CHX was found to result in significant reductions immediately after rinsing and at 60 minutes. Additionally, in an exploratory study with patients confirmed to be infected with SARS-CoV-2, the short-term efficacy of commercially available mouth rinses was compared to that of a negative control (rinsing with water) over a period of 2 hours.57Farmaha JK, James JN, Frazier K et al. Reduction of SARS-CoV-2 salivary viral load with pre-procedural mouth rinses: a randomised, controlled, clinical trial. Brit Dent J 2023;234(8):593-600. Compared to baseline, significant reductions in salivary viral loads were found immediately after rinsing with 0.12% CHX, 1.5% HP, 1% PVP-I or EO mouthrinses. A reduction at 2 hours was observed only for the groups rinsing with CHX or EO. In contrast, in another study no significant reductions in salivary viral loads were found at 30, 60, 120, or 180 minutes post-rinsing in comparison to the control group (no rinsing), nor any significant inter-group differences in one RCT involving rinsing with 0.2% CHX, 1.5% HP or CPC.58Perussolo J, Teh MT, Gkranias N et al. Efficacy of three antimicrobial mouthwashes in reducing SARS-CoV-2 viral load in the saliva of hospitalized patients: a randomized controlled pilot study. Sci Rep 2023;13(1):1647. doi: 10.1038/s41598-023-39308-x. In a second RCT with almost one hundred SARS-CoV-2-positive patients, salivary samples were obtained immediately prior to rinsing, as well as 30 minutes, 2, 4, 10 and 24 hours after rinsing with 0.05% CPC or 0.01% on-demand aqueous chlorine dioxide.59Onozuka D, Takatera S, Matsuo H et al. Oral mouthwashes for asymptomatic to mildly symptomatic adults with COVID-19 and salivary viral load: a randomized, placebo-controlled, open-label clinical trial. BMC Oral Health 2024;24(1):491. doi: 10.1186/s12903-024-04246-1. No significant differences in salivary viral load (CT values) were found in comparison to placebo at any time-point. Additionally, rinsing had been repeated at 4, 10 and 24 hours after salivary samples were obtained.

Similarly, no significant differences in salivary viral loads were found at any time point (30, 60 and 120 minutes) compared to baseline for test groups rinsing with 0.07% CPC, CHX, PVP-I, HP or distilled water (placebo) in another study.41Ferrer MD, Barrueco AS, Martinez-Beneyto Y et al. Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2. Sci Rep 2021;11(1):24392. https://doi.org/ 10.1038/s41598-021-03461-y. The researchers proposed that this could be due to lack of in vivo efficacy for the rinses, or that the viral RNA was detected but the particles were not infective, and also concluded that viral infectivity studies on the effect of mouthrinses are therefore required.41Ferrer MD, Barrueco AS, Martinez-Beneyto Y et al. Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2. Sci Rep 2021;11(1):24392. https://doi.org/ 10.1038/s41598-021-03461-y.

In other research, the antiviral efficacy of rinsing for 30 seconds with 20 ml of 0.075% CPC or 0.075% CPC plus 0.28% zinc lactate rinses was compared with distilled water (negative control).60Bezinelli LM, Corrêa L, Beyerstedt S et al. Reduction of SARS-CoV-2 viral load in saliva after rinsing with mouthwashes containing cetylpyridinium chloride: a randomized clinical study. PeerJ 2023;11:e15080. doi: 10.7717/peerj.15080. Salivary samples were taken at baseline, and 5, 30 and 60 minutes post-rinsing and then subjected to RT-qPCR testing, with amplification of the gene sequences for the N-protein and two others to determine salivary viral loads. The researchers found that the CPC+Zn rinse and CPC rinse, respectively, resulting in a 6.3 fold reduction and 2.5 fold reduction at 5 minutes, a 3.6 fold and 1.9 fold reduction at 30 minutes, and a 1.9 fold and 2 fold reduction at 60 minutes. (Table 1)

Table 1. In vivo studies using RT-PCR/RT-qPCR to assess anti-viral efficacy

In vivo research on anti-viral efficacy using other test methods

Several recent RCT have used test methods other than RT-PCR to evaluate mouthrinse efficacy. In one study, after baseline saliva samples were obtained, participants gargled with 20 ml of 1% HP for 30 seconds and saliva samples were again obtained 30 minutes post-rinsing.61Gottsauner MJ, Michaelides I, Schmidt B et al. A prospective clinical pilot study on the effects of a hydrogen peroxide mouthrinse on the intraoral viral load of SARS-CoV-2. Clin Oral Investig 2020;24(10):3707-13. Using RT-PCR testing, no significant reductions in viral load were found. In addition, no effect could be determined on infectivity since, based on cell cultures performed for samples with baseline viral loads of at least 103 RNA copies/mL, only one sample had yielded replicating virus.

In a second RCT using both RT-qPCR and cell cultures (viral cultures) as test methods, baseline saliva samples were obtained prior to participants rinsing with 2% PVP-I, 1% HP, 0.07% CPC or 0.12% CHX rinses, or distilled water (placebo).62Sánchez Barrueco Á, Mateos-Moreno MV, Martínez-Beneyto Y et al. Effect of oral antiseptics in reducing SARS-CoV-2 infectivity: evidence from a randomized double-blind clinical trial. Emerg Microbes Infect 2022;11(1):1833-42. doi: 10.1080/22221751.2022.2098059. While the RT-qPCR test indicated that no reductions in viral loads were obtained with any rinse, viral culture showed a significant effect for the 0.07% CPC rinse at 60 minutes, with a 97% reduction in viral infectivity compared to placebo.

In a third study, RT-qPCR, and tissue culture tests to determine the TCID50 were performed on oropharyngeal samples.32Bonn EL, Rohrhofer A, Audebert FX et al. Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2–Positive Patients: A Randomized Controlled Clinical Trial. J Dent Res 2023;102(6):608-15. doi:10.1177/00220345231156415. Participants gargled with 10 ml of 0.9% NaCl for 20 seconds, or the test rinse containing 0.05% CPC plus 0.05% CHX. Compared to baseline, a slight but significant reduction in viral load (RT-qPCR) was observed for the test group. Based on the second test method, a significant reduction in viral infectivity was found only for the test group (1.4 log₁₀ reduction in TCID50).

Evaluation of nucleocapsid protein (N-protein) levels has occurred in other studies. In a multi-center RCT, the researchers used RT-PCR testing, and developed and used the modified ELISA test to determine the level of N-proteins in samples.63Alemany A, Perez-Zsolt D, Raïch-Regué D et al. Cetylpyridinium Chloride Mouthwash to Reduce Shedding of Infectious SARS-CoV-2: A Double-Blind Randomized Clinical Trial. J Dent Res 2022;101(12):1450-6. doi: 10.1177/00220345221102310. Using these methods, the anti-viral efficacy of gargling for 1 minute with 15 ml of 0.07% CPC was compared to a negative control (colored distilled water) for oropharyngeal samples obtained 1 and 3 hours post-rinsing. Using the modified ELISA test, significantly higher levels of N-protein were observed in the test group at 1 hour and 3 hours, indicating destruction of the virus envelope (viral lysis), while with RT-PCR testing no significant difference in CT values (proxy for salivary viral loads) were found for the groups. In a second multicenter RCT using the same test methods to compare the effect of a 1-minute rinse with 0.07% CPC or placebo, samples were obtained at baseline and 2 hours post-rinsing.42Tarragó-Gil R, Gil-Mosteo MJ, Aza-Pascual-Salcedo M et al. Randomized clinical trial to assess the impact of oral intervention with cetylpyridinium chloride to reduce salivary SARS-CoV-2 viral load. J Clin Periodontol 2023;50(3):288-94. https://doi.org/10.1111/jcpe.13746 The modified ELISA test again showed significantly higher N-protein levels compared to placebo, confirming viral lysis and reduced infectivity, while the RT-PCR test again indicated no significant reduction in salivary viral load compared to placebo. (Table 2)

Table 2. In vivo studies using additional test methods

Conclusions

In vitro study results are supportive for the efficacy of CHX, CPC, EO and BAC against enveloped viruses. With respect to SARS-CoV-2, numerous in vitro and in vivo studies have been conducted to evaluate the anti-viral efficacy of antimicrobial rinses since the onset of the COVID-19 pandemic, with mixed results. Researchers have noted that studies were heterogeneous in study design, with small sample sizes and risks of bias, and that they were inconclusive.42Tarragó-Gil R, Gil-Mosteo MJ, Aza-Pascual-Salcedo M et al. Randomized clinical trial to assess the impact of oral intervention with cetylpyridinium chloride to reduce salivary SARS-CoV-2 viral load. J Clin Periodontol 2023;50(3):288-94. https://doi.org/10.1111/jcpe.13746^,48Ziaeefar P, Bostanghadiri N, Yousefzadeh P et al. The efficacy of mouthwashes in reducing SARS-CoV-2 viral loads in human saliva: A systematic review. New Microbes New Infect 2022;49:101064. doi: 10.1016/j.nmni.2022.101064.^,51Sbricoli L, Schiavon L, Brunello G et al. Efficacy of different mouthwashes against COVID-19: A systematic review and network meta-analysis. Jpn Dent Sci Rev 2023;59:334-56. doi: 10.1016/j.jdsr.2023.09.003.^,52Giulia B, Viktoria W, Robert K et al. Eligibility and efficacy of a CPC- and CHX-based antiviral mouthwash for the elimination of SARS-CoV-2 from the saliva: a randomized, double-blind, controlled clinical trial. J Clin Periodontol 2024;51(2):158-66. Further, different time points have been used for the collection of saliva samples, some of which may not be relevant for pre-procedural rinsing. Applicable research is needed with robust study designs and test methods, as well as research on the impact of post-rinsing viral load reductions on infectivity and transmission in the dental setting.

Nonetheless, the results of some studies are promising with respect to viral load reductions and alternative test methods have recently been used with promising results. While more research is needed, in the meantime, antimicrobial rinses can reduce antimicrobial loads and be used as an adjunct measure in the dental setting.

References

• 1.Marui VC, Souto MLS, Rovai ES et al. Efficacy of preprocedural mouthrinses in the reduction of microorganisms in aerosol: A systematic review. J Am Dent Assoc 2019;150(12):1015-26.e1. doi: 10.1016/j.adaj.2019.06.024.
• 2.Sreenivasan PK, Gittins E. The effects of a chlorhexidine mouthrinse on culturable micro-organisms of the tongue and saliva. Microbiol Res 2004;159(4):365-70.
• 3.Larsen PE. The effect of a chlorhexidine rinse on the incidence of alveolar osteitis following the surgical removal of impacted mandibular third molars. J Oral Maxillofac Surg 1991;49(9):932-37.
• 4.Hennessy B, Joyce A. A survey of preprocedural antiseptic mouth rinse use in Army dental clinics. Mil Med 2004;169(8):600-3.
• 5.American Dental Association. Interim Guidance for Minimizing Risk of COVID-19 Transmission. https://www.ada.org/~/media/CPS/Files/COVID/ADA_COVID_Int_Guidance_Treat_Pts.pdf.
• 6.Meng L, Fang H, Bian Z. Coronavirus Disease 2019 (COVID-19): Emerging and Future Challenges for Dental and Oral Medicine. J Dent Res 2020;99. 002203452091424. 10.1177/0022034520914246.
• 7.Peng X, Xu X, Li Y et al. Transmission routes of 2019-nCoV and controls in dental practice. Int J Oral Sci 2020;12:9.
• 8.Feres M, Figueiredo LC, Faveri M et al. The effectiveness of a preprocedural mouthrinse containing cetylpyridinium chloride in reducing bacteria in the dental office. J Am Dent Assoc 2010;141:415-22.
• 9.Domingo MA, Farrales MS, Loya RM et al. The effect of 1% povidone-iodine as a pre-procedural mouthrinse in 20 patients with varying degrees of oral hygiene. J Philipp Dent Assoc 1996;48(2):31-8.
• 10.DePaola LG, Eshenaur Spolarich A. Safety and Efficacy of Antimicrobial Mouthrinses in Clinical Practice. J Dent Hyg 2007;81(suppl 1) 117.
• 11.Scheie AA. Modes of action of currently known chemical antiplaque agents other than chlorhexidine. J Dent Res 1989;68 (Spec Iss):1609-16.
• 12.Nazzaro F, Fratianni F, De Martino L et al. Effect of essential oils on pathogenic bacteria. Pharmaceuticals (Basel) 2013;6(12):1451‐74. doi:10.3390/ph6121451.
• 13.Weber J, Bonn EL, Auer DL et al. Preprocedural mouthwashes for infection control in dentistry-an update. Clin Oral Investig 2023;27(Suppl 1):33-44. doi: 10.1007/s00784-023-04953-z.
• 14.Litsky BY, Mascis JD, Litsky W. Use of an antimicrobial mouthwash to minimize the bacterial aerosol contamination generated by the high-speed drill. PlumX Metrics. https://doi.org/10.1016/0030-4220(70)90407-X.
• 15.Cieplik F, Jakubovics NS. Preprocedural Mouthwashes for Reduction of SARS-CoV-2 Viral Load and Infectivity. J Dent Res 2022;101(12):1421-3. doi:10.1177/00220345221110444.
• 16.Muñoz-Basagoiti J, Perez-Zsolt D, León R et al. Mouthwashes with CPC Reduce the Infectivity of SARS-CoV-2 Variants In Vitro. J Dent Res 2021;100(11):1265-72. doi: 10.1177/00220345211029269.
• 17.Popkin DL, Zilka S, Dimaano M et al. Cetylpyridinium Chloride (CPC) Exhibits Potent, Rapid Activity Against Influenza Viruses in vitro and in vivo. Pathog Immun 2017;2(2):252-9. doi: 10.20411/pai.v2i2.200.
• 18.Dennison D K, Meredith G M, Shillitoe E J, Caffesse R G. The antiviral spectrum of Listerine antiseptic. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:442-8.
• 19.Kirk-Bailey J, Combes J, Sunkaraneni S, Challacombe S. The use of Povidone Iodine nasal spray and mouthwash during the current COVID-19 pandemic for the reduction of cross infection and protection of healthcare workers. https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3563092.
• 20.Centers for Disease Control and Prevention. Chemical Disinfectants Guideline for Disinfection and Sterilization in Healthcare Facilities (2008). https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-methods/chemical.html#Hydrogen.
• 21.Eggers M. Infectious Disease Management and Control with Povidone Iodine. Infect Dis Ther 2019;8:581-93.
• 22.Kanagalingam J, Feliciano R, Hah JH et al. Practical use of povidone-iodine antiseptic in the maintenance of oral health and in the prevention and treatment of common oropharyngeal infections. Int J Clin Pract 2015;69(11):1247-56. doi: 10.1111/ijcp.12707.
• 23.Mohd-Said S, Mohd-Dom TN, Suhaimi N et al. Effectiveness of Pre-procedural Mouth Rinses in Reducing Aerosol Contamination During Periodontal Prophylaxis: A Systematic Review. Front Med 2021;8:600769. doi: 10.3389/fmed.2021.600769.
• 24.Bernstein D, Schiff G, Echler G et al. In vitro Virucidal Effectiveness of a 0.12%-Chlorhexidine Gluconate Mouthrinse. J Dent Res 1990;69(3):874-876. doi:10.1177/00220345900690030901.
• 25.Riveira-Muñoz E, Garcia-Vidal E, Bañó-Polo M et al. Cetylpyridinium Chloride-Containing Mouthwashes Show Virucidal Activity against Herpes Simplex Virus Type 1. Viruses 2023;15:1433. https://doi.org/10.3390/v15071433.
• 26.Kawana R, Kitamura T, Nakagomi O et al. Inactivation of Human Viruses by Povidone-Iodine in Comparison with Other Antiseptics. Dermatol 1997;195 (Suppl. 2):29-5. https://doi.org/10.1159/000246027
• 27.Meiller TF, Silva A, Ferreira SM et al. Efficacy of Listerine® Antiseptic in reducing viral contamination of saliva. J Clin Periodontol 2005;32:341-6. https://doi.org/10.1111/j.1600-051X.2005.00673.x.
• 28.Welch SR, Davies KA, Buczkowski H et al. Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives. J Clin Microbiol 2020;58(11):e01713-20. doi: 10.1128/JCM.01713-20.
• 29.Hong W, Xiong J, Nyaruaba R et al. Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. Sci Total Environ 2021;797:149085. doi: 10.1016/j.scitotenv.2021.149085.
• 30.Fernandez MDS, Guedes MIF, Langa GPJ et al. Virucidal efficacy of chlorhexidine: a systematic review. Odontol 2022;110(2):376-92. doi: 10.1007/s10266-021-00660-x.
• 31.Moretti M. Viral Titer Quantification: Balancing Traditional and Modern Approaches for Comprehensive Analysis. Dec 26, 2023. https://www.labmanager.com/viral-titer-quantification-balancing-traditional-and-modern-approaches-for-comprehensive-analysis-31559.
• 32.Bonn EL, Rohrhofer A, Audebert FX et al. Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2–Positive Patients: A Randomized Controlled Clinical Trial. J Dent Res 2023;102(6):608-15. doi:10.1177/00220345231156415.
• 33.Taylor A, Calvez R, Atkins M, Fink CG. Comparing lateral flow testing with a rapid RT-PCR method for SARS-CoV-2 detection in the United Kingdom-A retrospective diagnostic accuracy study. Health Sci Rep 2022;5(5):e811. doi: 10.1002/hsr2.811.
• 34.What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR? https://www.enzo.com/note/what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-qpcr/.
• 35.Finks SW, Van Matre E, Budd W et al. Clinical Significance of Quantitative Viral Load in Patients Positive for SARS-CoV-2. Am J Med Open 2023;10:100050. doi: 10.1016/j.ajmo.2023.100050.
• 36.Michalakis Y, Sofonea MT, Alizon S, Bravo IG. SARS-CoV-2 viral RNA levels are not 'viral load'. Trends Microbiol 2021;29(11):970-2. doi: 10.1016/j.tim.2021.08.008.
• 37.Park J, Cho SI, Kang SG et al. Long-term trends in cycle threshold values: a comprehensive analysis of COVID-19 dynamics, viral load, and reproduction number in South Korea. Front Public Health 2024;12:1394565. doi: 10.3389/fpubh.2024.1394565.
• 38.Bhavnani D, James ER, Johnson KE et al. SARS-CoV-2 viral load is associated with risk of transmission to household and community contacts. BMC Infect Dis 2022;22(1):672. doi: 10.1186/s12879-022-07663-1.
• 39.Lee LYW, Rozmanowski S, Pang M et al. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infectivity by Viral Load, S Gene Variants and Demographic Factors, and the Utility of Lateral Flow Devices to Prevent Transmission. Clin Infect Dis 2022;74(3):407-15. doi: 10.1093/cid/ciab421.
• 40.Harris T, Geard N, Zachreson C. Correlation of viral loads in disease transmission could affect early estimates of the reproduction number. J R Soc Interface 2023;20(202):20220827. doi: 10.1098/rsif.2022.0827.
• 41.Ferrer MD, Barrueco AS, Martinez-Beneyto Y et al. Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2. Sci Rep 2021;11(1):24392. https://doi.org/ 10.1038/s41598-021-03461-y.
• 42.Tarragó-Gil R, Gil-Mosteo MJ, Aza-Pascual-Salcedo M et al. Randomized clinical trial to assess the impact of oral intervention with cetylpyridinium chloride to reduce salivary SARS-CoV-2 viral load. J Clin Periodontol 2023;50(3):288-94. https://doi.org/10.1111/jcpe.13746
• 43.Mezarina Mendoza JPI, Trelles Ubillús BP, Salcedo Bolívar GT et al. Antiviral effect of mouthwashes against SARS-COV-2: A systematic review. Saudi Dent J 2022;34(3):167-93. doi: 10.1016/j.sdentj.2022.01.006.
• 44.Xu C, Wang A, Hoskin ER et al. Differential Effects of Antiseptic Mouth Rinses on SARS-CoV-2 Infectivity In Vitro. Pathogens 2021;10(3):272. https://doi.org/10.3390/pathogens10030272.
• 45.Meister TL, Brüggemann Y, Todt D et al. Virucidal Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome Coronavirus 2. J Infect Dis 2020;222(8):1289-92. https://doi.org/10.1093/infdis/jiaa471.
• 46.Ebrahimi T, Shamshiri AR, Alebouyeh M, Mohebbi SZ. Effectiveness of mouthwashes on reducing SARS-CoV-2 viral load in oral cavity: a systematic review and meta-analysis. BMC Oral Health 2023;23(1):443.
• 47.Lin SY, Sun JS, Hung MC, Chang JZ. Effectiveness of mouth rinses against COVID-19: a systematic review and network meta-analysis. J Hosp Infect 2023;139:175-91. doi: 10.1016/j.jhin.2023.06.022.
• 48.Ziaeefar P, Bostanghadiri N, Yousefzadeh P et al. The efficacy of mouthwashes in reducing SARS-CoV-2 viral loads in human saliva: A systematic review. New Microbes New Infect 2022;49:101064. doi: 10.1016/j.nmni.2022.101064.
• 49.Zhang M, Meng N, Duo H et al. Efficacy of mouthwash on reducing salivary SARS-CoV-2 viral load and clinical symptoms: a systematic review and meta-analysis. BMC Infect Dis 2023;23(1):678. doi: 10.1186/s12879-023-08669-z.
• 50.D'Amico F, Moro M, Saracino M et al. Efficacy of Cetylpyridinium Chloride mouthwash against SARS-CoV-2: A systematic review of randomized controlled trials. Mol Oral Microbiol 2023;38(3):171-80. doi: 10.1111/omi.12408.
• 51.Sbricoli L, Schiavon L, Brunello G et al. Efficacy of different mouthwashes against COVID-19: A systematic review and network meta-analysis. Jpn Dent Sci Rev 2023;59:334-56. doi: 10.1016/j.jdsr.2023.09.003.
• 52.Giulia B, Viktoria W, Robert K et al. Eligibility and efficacy of a CPC- and CHX-based antiviral mouthwash for the elimination of SARS-CoV-2 from the saliva: a randomized, double-blind, controlled clinical trial. J Clin Periodontol 2024;51(2):158-66.
• 53.Alzahrani MM, Bamashmous S, Alkharobi H et al. Mouth rinses efficacy on salivary SARS-CoV-2 viral load: A randomized clinical trial. J Med Virol 2023;95(1):e28412. doi: 10.1002/jmv.28412.
• 54.Seneviratne CJ, Balan P, Ko KKK et al. Efficacy of commercial mouth-rinses on SARS-CoV-2 viral load in saliva: randomized control trial in Singapore. Infection 2021;49:305-11. https://doi.org/10.1007/s15010-020-01563-9.
• 55.Chaudhary P, Melkonyan A, Meethil A et al. Estimating salivary carriage of severe acute respiratory syndrome coronavirus 2 in nonsymptomatic people and efficacy of mouthrinse in reducing viral load: A randomized controlled trial. J Am Dent Assoc 2021;152(11):903-8. doi: 10.1016/j.adaj.2021.05.021.
• 56.Eduardo FP, Corrêa L, Heller D, et al. Salivary SARS-CoV-2 load reduction with mouthwash use: A randomized pilot clinical trial. Heliyon 2021;7(6):e07346.
• 57.Farmaha JK, James JN, Frazier K et al. Reduction of SARS-CoV-2 salivary viral load with pre-procedural mouth rinses: a randomised, controlled, clinical trial. Brit Dent J 2023;234(8):593-600.
• 58.Perussolo J, Teh MT, Gkranias N et al. Efficacy of three antimicrobial mouthwashes in reducing SARS-CoV-2 viral load in the saliva of hospitalized patients: a randomized controlled pilot study. Sci Rep 2023;13(1):1647. doi: 10.1038/s41598-023-39308-x.
• 59.Onozuka D, Takatera S, Matsuo H et al. Oral mouthwashes for asymptomatic to mildly symptomatic adults with COVID-19 and salivary viral load: a randomized, placebo-controlled, open-label clinical trial. BMC Oral Health 2024;24(1):491. doi: 10.1186/s12903-024-04246-1.
• 60.Bezinelli LM, Corrêa L, Beyerstedt S et al. Reduction of SARS-CoV-2 viral load in saliva after rinsing with mouthwashes containing cetylpyridinium chloride: a randomized clinical study. PeerJ 2023;11:e15080. doi: 10.7717/peerj.15080.
• 61.Gottsauner MJ, Michaelides I, Schmidt B et al. A prospective clinical pilot study on the effects of a hydrogen peroxide mouthrinse on the intraoral viral load of SARS-CoV-2. Clin Oral Investig 2020;24(10):3707-13.
• 62.Sánchez Barrueco Á, Mateos-Moreno MV, Martínez-Beneyto Y et al. Effect of oral antiseptics in reducing SARS-CoV-2 infectivity: evidence from a randomized double-blind clinical trial. Emerg Microbes Infect 2022;11(1):1833-42. doi: 10.1080/22221751.2022.2098059.
• 63.Alemany A, Perez-Zsolt D, Raïch-Regué D et al. Cetylpyridinium Chloride Mouthwash to Reduce Shedding of Infectious SARS-CoV-2: A Double-Blind Randomized Clinical Trial. J Dent Res 2022;101(12):1450-6. doi: 10.1177/00220345221102310.