key: cord-0858556-lg8uusbx
authors: Tadakamadla, Jyothi; Boccalari, Elisa; Rathore, Vatsna; Dolci, Carolina; Tartaglia, Gianluca M.; Tadakamadla, Santosh Kumar
title: In vitro studies evaluating the efficacy of mouth rinses on Sars-Cov-2: a systematic review
date: 2021-08-05
journal: J Infect Public Health
DOI: 10.1016/j.jiph.2021.07.020
sha: 54440902cd4d43b08cdbdbe19ad0f64ed82ebbc0
doc_id: 858556
cord_uid: lg8uusbx

This systematic review aims to evaluate the efficacy of several mouth rinses on SARS-CoV-2 from in vitro studies. Five electronic databases were searched up to February 2021; no language or time restrictions were used. Two independent reviewers conducted both selection and data extraction processes. The toxicological data reliability assessment tool was used to evaluate the risk of bias. Starting from 239 articles, retrieved by the electronic search, only eight studies were included in our systematic review. Povidone Iodine (PVP-I) showed its efficacy in killing SARS-CoV-2, demonstrating higher virucidal activity than other commonly used molecules. Conflicting results were found about the effectiveness of Chlorhexidine (CHX) while hydrogen peroxide (H(2)O(2)) proved less effective than PVP-I. Other actives, such as quaternary ammonium compounds and Ethanol (particularly when combined with essential oils), have also shown promising results in reducing viral load, with results comparable to PVP-I.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a Betacoronavirus linked to the pneumonia outbreak firstly discovered in Wuhan, Hubei province, China, in late 2019 which triggered a pandemic and health crisis. It is mainly transmitted human to human through droplets (small particles >5 µm produced while coughing, sneezing or even speaking), contact with an infected subject or direct or indirect contact with contaminated surfaces [1, 2] . Its aerosol transmission has been studied, too, and it occurs primarily in health care facilities through aerosol-generating procedures (AGPs) [2] . AGPs include extubation, intubation, tracheotomy, positive pressure ventilation (CPAP), bronchoscopy and cardiopulmonary resuscitation [3] . High risk is also associated with dental procedures, such as the use of ultrasonic devices, 3-in-1 air-water syringes and high-speed handpieces [4] .

According to Xu et al., the primary host cell receptor for SARS-CoV-2 is the angiotensin converting enzyme II (ACE2), which binds with the viral spike protein; epithelial cells of the oral mucosa have a high expression of ACE2 [5] . The presence of a high viral load has been confirmed in the saliva of symptomatic patients with coronavirus disease (COVID-19) with a higher viral load in the first week after the onset of symptom, which then starts to decrease with time [6, 7] . Moreover, even in the oropharynx of asymptomatic patients, high viral load has been identified, which emphasises the prominent role of the oral cavity in the transmission of the virus [8] .

Therefore, dental professionals are at high risk of SARS-CoV-2 infection because of their proximity to patients and AGP [9] . Since the onset of the pandemic, the scientific community has been focusing on preventing transmission of SARS-CoV-2 infection by using personal protective equipment, limiting the practice of AGPs, and using the rubber dam. With the emerging evidence on the virucidal J o u r n a l P r e -p r o o f efficacy of mouth rinse molecules, pre-procedural mouth rinsing is being advocated by several regulatory organisations throughout the world, including the American Centers for Disease Control and Prevention, to reduce SARS-CoV-2 transmission in a dental setting [4] . There are many different molecules already effectively used against bacteria or viruses and currently being studied for their potential virucidal effect on SARS-CoV-2. Several studies in vitro and a few in vivo have been conducted on the efficacy of various mouthwashes to reduce viral load, and thus the spread of the infection, before performing any intraoral procedures.

There have been several attempts to conduct systematic reviews on this topic. For instance, the Cochrane collaboration has commissioned three systematic reviews, but none of these could find any robust studies exploring the effectiveness of mouth rinses or nasal sprays on COVID-19 viral load [10] [11] [12] . There are several in vivo studies still ongoing with very little evidence available from them.

Our systematic review aims to evaluate the current literature about the effectiveness of different mouthwash actives on SARS-CoV-2 in vitro.

This review conforms to the preferred reporting items for systematic reviews (PRISMA) reporting guidelines [13] .

The PICO question of this review was what is the virucidal activity (outcome) of different mouth rinse molecules (intervention and comparison) on the SARS-COV-2 virus (participants)? Any invitro study evaluating the effectiveness of any mouth rinse on SARS-COV2 viral load or any other related outcome were considered for inclusion. All clinical investigations were excluded. Besides, reviews, opinion pieces, perspectives, conference papers, book chapter, abstracts were excluded. We also reviewed the cross-references of the included articles. Any study evaluating the effectiveness of a mouth rinse molecule or new formulation in the form of a mouth rinse on SARS-COV-2 virus from any source was considered for inclusion.

Five databases were searched on 9 th February 2021 from inception, which included Medline via Pubmed, Cinahl, Embase, Cochrane and Web of science. The search strategy involved two strings of J o u r n a l P r e -p r o o f keywords combined by the Boolean operator 'AND'. "Mouth wash OR mouthwash OR mouth rinse OR mouth rinse OR oral rinse OR rinse" AND "Betacoronavirus OR SARS CoV 2 OR SARSCoV2 OR SARS-CoV2 OR Coronavirus Infections OR COVID-19 Virus OR COVID-19 OR Coronavirus OR Severe Acute Respiratory Syndrome Coronavirus 2 OR 2019nCoV". A mix of medical subject headings and free keywords were used. No language or time limiters were used. The search strategy was implemented and tested by two reviewers independently (JT and SKT); no discrepancy was found between the reviewers.

All the retrieved titles and abstracts were exported to a referencing software program (EndNote X9, Philadelphia, Clarivate). Any duplicates found were deleted. Two reviewers (JT and SKT) screened all the titles and abstracts independently; those that seemed suitable were considered for inclusion in the full-text review. When the information provided in the abstract and title were inadequate to determine eligibility, articles were included in the full-text review. Any discrepancy between the reviewers (SKT and JT) was resolved by discussion. A pre-piloted data extraction chart was used to extract the data from the included studies, which was done independently by two reviewers (JT and VR). All the extracted data was checked for accuracy by a third reviewer (SKT). The pre-piloted charts had data fields on Author, year, country of origin or study setting, source of SARS-COV-2 virus, active ingredient or mouth rinse molecule, formulation, concentration, contact time, comparison groups, outcomes assessed, method of evaluating the outcome, findings and overall conclusions.

A quantitative analysis was not feasible due to the heterogeneity in the molecules tested and the contact times used. Therefore, the included studies' background characteristics, the molecules they tested, and their outcomes are reported qualitatively. Findings have been organised according to the mouth rinse molecule tested.

The Risk of Bias Assessment was done using the Toxicological data reliability assessment tool (TOXRTOOL) [14] . For in vitro studies, it uses a set of 18 questions marking 1 or 0 against each criterion and simple arithmetic summation of all values to categorise as 1, 2, or 3. If the value is ≥15, J o u r n a l P r e -p r o o f category one is assigned. For values >11, category 2 is assigned, and for all values < 11, category 3 is assigned. Categories 1 and 2 represent that the data is reliable without and with restriction, respectively, while category 3 indicates that the data reported from the study is not reliable.

A total of 239 titles were retrieved from the electronic search; 162 remained after removal of duplicates and were screened by titles and abstracts (Figure 1 ). Thirteen articles were found relevant for a full-text review, with eight articles being included in this review. Five articles were excluded, three assessed the effectiveness of mouth rinses on viruses other than SARS-COV-2 [15] [16] [17] , one each was an in-silico study [18] and an opinion piece [19] .

General characteristics of the included studies are presented in Table 1 . Eight in vitro studies were included in this systematic review. Four studies were conducted in the USA [20] [21] [22] [23] with one each in Malaysia [24] , Singapore [25] , Germany [26] , and India [27] . In all studies, the outcome assessed was the virucidal activity of the active ingredient against SARS CoV-2. The typical active ingredient tested in all studies was Povidone Iodine (PVP-I). While five studies [20-22, 24, 25] assessed Povidone Iodine (PVP-I) at different concentrations and formulations, the other three [23, 26, 27] assessed PVP along with other active ingredients, such as chlorhexidine, hydrogen peroxide, and Ethanol. The source of virus in the studies has varied according to the country of origin, while those from the USA have used USA-WA1/2020 strain, the study from Malaysia used SARS-COV-2/MY/UM/6-3;TIDREC [24] . Anderson et al. [25] from Singapore used hCoV-19/Singapore/2/2020 while Meister et al. [26] used BetaCoV/Germany/Ulm/01/2020 and BetaCoV/Germany/Ulm/02/2020. Throat swabs from an infected patient were also used by Jain et.al [27] and Meisteret.al [26] . The contact time in the studies ranged from as low as 15 seconds to 30

min. The most commonly tested contact time was 30 seconds, used in 6 studies [20, 21, [24] [25] [26] [27] . The lowest contact time tested was 15 seconds, used by 3 studies [20, 21, 24] and the longest contact time of 30 minutes was tested by Xu et.al [23] . formulation [20] [21] [22] 25] . One study observed that the log reduction factor differed based on the viral strains, with >3.11, >2.78 and >2.61 being reported for strain 1, 2 and 3, respectively [26] . Among the reviewed studies, a kill rate of > 5 log 10 was reported at 15 seconds (1% PVP) and 30 seconds (0.5% PVP) by one study [24] > 4 log 10 at 15 [20, 24] and 60 seconds [22, 27] was observed by two studies, while Anderson et al., also reported a kill rate of > 4 log 10 at 30 seconds [25] . Three studies observed a kill rate of > 3log 10 even at short contact times of 15 and 30 seconds [20, 21, 27] .

Virucidal activity of >99.9% was found by one study that tested a contact time of 30 minutes [23] .

Two studies assessed the virucidal activity of H2O2 [23, 26] , formulated as a mouthwash. [23] . One study compared Hydrogen peroxide's effectiveness with PVP-I and found PVP-I to be more effective [20] .

The CHX mouthwash was assessed by three studies [23, 26, 27] . One study assessed the effectiveness of CHX concentration at 0.2% and 0.12% [27] , while another tested 0.12% concentration in comparison to other actives [23] . On the other hand, the third study tested two different concentrations but did not report the concentration of one of the products used in their study [26] . 

A 70% solution of Ethanol was used as positive control by three studies and had PVP-I has their active, and they found Ethanol to be as effective as PVP-I [20] [21] [22] .

Besides the above described active ingredients, essential oils were assessed in two studies by testing commercially available mouth rinses (i.e. Listerine, Listerine ultra) and proved to be as effective as PVP-I in reducing viral titre [23] [26, 28] . A mouth rinse with Dequalinium chloride/benzalkonium chloride, two quaternary ammonium compounds, achieved a log reduction value of ≥3.11, ≥2.78 and ≥2.61 on the three different strains, thus mirroring the results of PVP-I., as reported by Meister et al. [26] . Studies also tested compounds such as Sodium Bicarbonate, Sodium chloride, Octenidine dihydrochloride, but in general, they had inferior virucidal activity compared to PVP-I.

In this review, six studies were found to have an overall score of ≥15 (category 1) demonstrating that data from these studies is reliable without restrictions. Two studies had a score of 14 and belonged to category 2. None of the studies had unreliable data (category 3). The scoring for all studies is shown in Table 2 .

This systematic review attempted to provide synthesised evidence on the efficacy of different mouth rinse molecules on the SARS-COV-2 virus and found promising results with PVP-I demonstrating better efficacy than other tested mouth rinses. The results indicate that pre-procedural mouth rinses could be a significant measure to prevent viral spread in the dental office. [29] . All the evaluated in vitro studies considered PVP-I, highlighting its reduction potential in inactivating SARS-CoV-2 even at 15 seconds time contact and at low concentrations. However, cytotoxic effects of PVP-I must be taken into consideration since it is toxic for the oral and nasal mucosa in a concentration higher than 2.5% and 5%, respectively [30] [21] . Even though commercial formulations do not reach those concentrations, cytotoxicity is an important parameter to evaluate that can influence the mouth rinse's effectiveness and must be taken into account [23] . Moreover, PVP-I mouth rinse cannot be used in pregnant women, subjects allergic to iodine, and patients with underlying thyroid disease or undergoing radioactive iodine therapy [21] .

and orthomyxoviruses), is known to be highly sensitive to a high concentration of Ethanol (60%-70%) used in surface decontamination since it interferes with the lipid envelope and causes the virus inactivation [31] . Commercial mouth rinses have a lower concentration of Ethanol. Still, studies proved them to be effective against different enveloped viruses (other than SARS-CoV-2) even at lower concentration (21%-27%) when combined with essential oils [32] [33] . Ethanol was assessed in five examined studies; three used it as a positive control in high concentration (70%), while the remaining studies used Ethanol in a formulation with essential oils. Notably, this last combination proved to be effective, similarly to PVP-I, in reducing SARS-CoV-s viral titre and with less cytotoxicity [23] [26] . Other actives, such as quaternary ammonium compounds like dequalinium chloride and benzalkonium chloride, were also assessed in the evaluated studies and were able to reduce SARS-COV-2 viral titre to undetectable levels even though, here too, the complex mechanism behind viral inactivation is still not clear [26] [23] .

Another common molecule under evaluation is H202, with its known ability to inactivate influenza A and B viruses, adenoviruses and rhinoviruses, causing nucleic acid damages and increasing cell membrane permeability [7] . Despite these previous findings, H2O2 did not demonstrate great capacity in reducing viral titre [26] [20] . Moreover, hydrogen peroxide proved to be more cytotoxic than PVP-I, even if diluted [23] .

CHX is the most widely used active in mouthwashes for its bactericidal and bacteriostatic effects, thanks to its positive charge that allows it to interact with the microbial surface, which is negatively charged. In particular, it is effective against an enveloped viruses, such as Influenza A, herpesvirus 1, cytomegalovirus [7, 28, 31] . The results from studies that tested CHX were mixed. While two studies found CHX to have an efficacy better than PVP-I or comparable with other actives, one study found it to perform poorly than other active (PVP-I, Ethanol+essential oils and Dequalinium chloride+benzalkonium chloride) [23, 26, 27] .

Despite most studies demonstrating a low risk of bias, this systematic review's results are limited because of several methodological aspects and comparison groups varied between the studies. The findings support the scientific rationale for proposing pre-procedural mouthrinses based on the evidence gathered from in vitro studies. We anticipate that randomised controlled trials with a larger sample size will support or refute the findings of this review. Several clinical trials have already been registered, while a few articles with preliminary results have already been published [34] [35] [36] .

In conclusion, PVP-I was the most widely tested active ingredient. Evidence from in vitro studies demonstrates that PVP-I has higher virucidal activity than other commonly used molecules. Although very few studies tested the effectiveness of dequalinium chloride+benzalkonium chloride and Ethanol (alone or with essential oils), they effectively reduced viral load comparable to PVP-I. Findings related to CHX were contrasting between the studies, while H202 was found to have inferior properties compared to other widely used mouth rinse molecules. However, caution is required as this finding comes from only one study. Clinical studies will be essential to confirm the effectiveness of mouth rinse molecules discussed in this review.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

None declared

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