IN VITRO ANTI-VIRAL ACTIVITY OF HEXETIDINE (BACTIDOL®) ORAL MOUTHWASH AGAINST HUMAN CORONAVIRUS OC43 AND INFLUENZA A (H1N1) VIRUS

Mouthwashes are used to decrease oral cavity microbial load due to their antiseptic properties. Hexetidine is a broad-spectrum antiseptic used for minor infections of mucous membranes, and in particular as a 0.1% mouthwash for local infections and oral hygiene. This study determined the anti-viral activity of the mouthwash hexetidine (Bactidol®), specifically in reducing viral concentration of Human Coronavirus OC43 (HCoV OC43; ATCC® VR-1558™) and Influenza A virus (IAV H1N1; clinical strain) in Vero 6 and MDCK cell cultures respectively, using in-vitro suspension assay (ASTM E-1052-11) designed to evaluate virucidal property of microbicides like hexetidine. Study results indicated that hexetidine was able to reduce infectivity of HCoV OC43 and IAV H1N1 at 25%, 50% and 100% concentrations by more than 80% at 15- and 30-seconds exposure times. One hundred percent (100%) concentration of hexetidine was found to be cytotoxic to MDCK cell line used for IAV H1N1 propagation. Hexetidine-treated cell lines achieved >80% survival rate for MDCK and Vero E6 at a contact time of 15 seconds and 30 seconds (which are the approximate times of gargling with hexetidine mouthwash). The anti-viral activity of hexetidine mouthwash against other more virulent or pathogenic coronaviruses like SARS-CoV-2 can be explored further.


INTRODUCTION
Mouthwashes have been widely used in oral hygiene. Most of the mouthwashes are used to decrease oral cavity microbial load due to their anti-septic properties and have been prescribed in dentistry. The active ingredients of mouthwashes, among others are hexetidine, benzydamine hydrochloride, and povidone iodine.
Hexetidine belongs to the group of pyrimidine derivatives. It is a broad-spectrum antiseptic, active in vitro and in vivo against Gram-positive and Gram-negative bacteria. It is used for minor infections of mucous membranes, and in particular as a 0.1% mouthwash for local infections and oral hygiene. Pharmacological evidence indicated that the primary mode of action is due to its interference with vital metabolic processes necessary for the growth of microorganisms [1].
Povidone iodine is an iodophore that is used as a disinfectant and antiseptic. Iodophores are loose complexes of iodine and carrier polymers. Solutions of povidone-iodine gradually release iodine to exert an effect against bacteria, fungi, viruses, protozoa, cysts, and spores. A 1% mouthwash has been used for oral infections including candidiasis [1].
Antimicrobial efficacy may be measured directly by the effect of certain compounds on the growth of microorganisms in vitro, or indirectly by studying their effect on certain health conditions that are caused or affected by microorganisms. Some of these conditions are halitosis, plaque, gingivitis and periodontitis. [1] From the literature review above [2], hexetidine and povidone iodine generally had good efficacy against the bacterial flora of the oral cavity, with hexetidine being slightly superior between the two. In addition, hexetidine lost only approximately 25% of its antimicrobial action after 60 minutes while povidone iodine lost almost 40% of its antimicrobial efficacy after 10 minutes [3].
The sustained antimicrobial efficacy of hexetidine was also noted in another study [4]; however, it was noted that the duration was shorter than for chlorhexidine [3,4]. Hexetidine was also found to have good efficacy against plaque and gingivitis [5,6] but was again inferior to chlorhexidine [7].
Four other studies found that the antimicrobial action of povidone iodine had a short duration [3,[11][12][13]. Some studies, however, found povidone iodine had higher antimicrobial efficacy than chlorhexidine [14,15]. Povidone iodine was also noted to reduce Streptococcus mutans count although not to the same degree as chlorhexidine [16]. It was more effective in alleviating mucositis than chlorhexidine [8] and was as effective as chlorhexidine for plaque and gingivitis [17].
The literature review above [2] concluded based on the in vitro, in vivo and clinical studies discussed, which used both direct and indirect methods to measure antimicrobial efficacy, that hexetidine, chlorhexidine and povidone-iodine were effective against oral microbial flora and differ primarily in the duration of their antimicrobial action.
Zoonotic coronaviruses were discovered in the 1960s and pathogenic human coronaviruses were discovered in 2002. Currently, there are now seven human coronaviruses which include the SARS-CoV-2. Most of these human coronaviruses cause mild diseases, and these include the HCoVs OC43, 229E, NL63 and HKU1. The more virulent species are SARS-CoV, MERS-CoV and SARS-CoV-2. Coronaviruses are positive sense, single stranded RNA virus, spherical and enveloped with club-shaped spikes on the surface looking like the solar corona. The four coronaviruses genera are α, β, γ, δ. The human α-CoV are HCoV-229E and NL63, while the human β-CoV are MERS-CoV, SARS-CoV, HCoV OC43 and HCoV-HKU1. SARS-CoV-2 and HCoV OC43 are both β-CoV. [18] This study determined the anti-viral activity of hexetidine (Bactidol®), specifically in reducing viral concentration of Human Coronavirus (HCoV OC43) and Influenza A virus (IAV H1N1) infected cell cultures by demonstrating its Tissue Culture Infective Dose or TCID50/ml in 25%, 50% and 100% aqueous hexetidine concentration at 15-and 30-seconds exposure times (which are the approximate times of gargling with Bactidol® mouthwash).

MATERIALS and METHODS
The study was done from 10 September 2020 to 03 November 2020. The test method used was according to the American Society for Testing and Materials International -ASTM E-1052-11 Standard test method, which assess the activity of microbicides against viruses in suspension (2011) [19].

Tissue culture cells. Madin Darby Canine Kidney (MDCK) cells from WHO Collaborating
Centre for Reference and Research on Influenza, Australia, and African green monkey kidney Vero E6 cells from the Department of Virology, Tohoku University Graduate School of Medicine, Sendai, Japan were used. These cell lines are permissive and susceptible for the test viruses IAV H1N1 and HCoV OC43, respectively. These cultures were maintained in tissue culture media with additives.
Test viruses. Influenza A virus H1N1 (IAV H1N1), came from a clinical sample from Influenza Surveillance, were grown in MDCK cells, confirmed by Hemagglutination Inhibition and RT-PCR. Human Coronavirus (HCoV FR-302), Strain OC43 (ATCC ® VR-1558™) was purchased from American Type Culture Collection (ATCC). These are the common respiratory viruses that can cause sore throat and are found in the oral cavity; HCoV is a β-CoV similar to SARS-CoV-2. These viruses were grown on viral culture media with additives (L-glutamine, Penicillin-Streptomycin). Antiviral agents. Hexetidine (Bactidol®) at 100% (undiluted), aqueous 50% and 25% concentrations were used. 70% ethyl alcohol served as a positive control.
The method used was an in-vitro suspension assay designed to evaluate virucidal property of the product against IAV H1N1 and HCoV OC43. Presence of infective virus was determined via monitoring of cytopathic effect (CPE) on the appropriate cell line. A 0.2 ml suspension of virus was exposed to 1.8mI per specified concentration of anti-viral test agents. At each specified exposure time, an aliquot was removed and neutralized by serial dilution and assayed for presence of virus. Virus controls, cytotoxicity controls, and neutralization controls were assayed in parallel.
After incubation, virus infected cell cultures in wells were identified through their characteristic CPE, rounding of cells (MDCK) and vacuolization of the cytoplasm and sloughing of cells (Vero E6). Recorded results were used to calculate infecting activity (TCID50) through Spearman-Karber method [20]. Percent reduction and log reduction were subsequently computed.
All tests were done at the Virology Department of the Research Institute for Tropical Medicine (RITM), Department of Health. The Institutional Review Board (IRB) of RITM was accordingly informed of this basic laboratory study involving viral cell cultures and does not require to undergo review as per regulations on studies involving animals or humans as test subjects.
However, one hundred percent (100%) concentration of hexetidine (Bactidol®) was found to be cytotoxic to the MDCK cell line used for IAV H1N1 propagation. The following figures were taken during the study, Hexetidine (Bactidol®) mouthwash showed inhibition of the virus induced CPE in the infected MDCK and Vero 6 cells, Figure 1. letters A and C, respectively.

DISCUSSION
This study showed that hexetidine (Bactidol®) was able to reduce infectivity of HCoV OC43 (ATCC ® VR-1558™) and IAV H1N1 at 25%, 50% and 100% concentrations. These hexetidine concentrations reduced by more than 94% to 99% the infectivity of Human Coronavirus OC43 at 15 and 30 seconds exposure times. Antiseptic mouthwashes have been widely used as a standard measure before routine dental treatment, especially pre-operatively [22,23]. These are widely used solutions for rinsing the mouth due to their ability to reduce the number of microorganisms in the oral cavity and colonyforming units in dental aerosols [24]. The use of mouthwashes and gargles were deemed to be relatively safe, with only minor adverse effects with long-term use [25].
Vergara-Buenaventura A and Castro-Ruiz C (2020) suggested the use of pre-procedural mouthwashes in dental practice to reduce SARS-CoV-2 viral load from dental procedures and to reduce the cross-infection risk while treating patients during the pandemic (26).
The SARS-CoV-2 spike protein in the membrane envelope is a typical structure of coronaviruses [26][27][28]. The spike protein interacts with the angiotensin-converting enzyme 2 (ACE2) receptors of the host cells enabling the virus to enter the cells [29]. Different tissue cells, including those of the mucosal tissues, gingiva, periodontal pockets, tongue and salivary glands present possible infection routes as these have membranes bound to ACE2 [29][30][31][32][33][34]. Such oral tissues are sources from which SARS-CoV-2 transmission may occur during dental care, talking, coughing, and sneezing (35,36).
The American Dental Association [37] and the Center for Disease Control and Prevention [38] have recommended the use of pre-procedural mouthwashes before oral procedures, despite no clinical evidence that the use of mouthwashes could prevent SARS-CoV-2 transmission. [26] In the Philippines, among the active agents used in commercially available mouthwashes, only povidone-iodine had been shown to have antiviral activity in vitro against SARSCoV-2 [39].
This in vitro study showed that hexetidine, even at diluted concentrations reduced the infectivity of the two oral virus strains, HCoV OC43 and Influenza A virus H1N1 when used for 15 and 30 seconds. The anti-viral activity of hexetidine mouthwash against the other more virulent members of the Coronavirus Family, SARS-CoV-2 can be explored using the methods used in this in vitro study. Clinical studies can also follow, to evaluate the effectiveness of antiseptic mouthwashes like hexetidine on SARS-CoV-2, the subjects may be with asymptomatic or mildly infected individuals. The future study will determine whether hexetidine (Bactidol®) can lessen viral load or shorten the duration of viral carriage.

ACKNOWLEDGEMENT
Thanks is due to the Research Institute for Tropical Medicine-Department of Health and its Virology Department Staff Mayan U Lumandas, Vina Lea F Arguelles, Ma Therese DC Quimpo, and Janiza Ianne M Foronda, who did the laboratory tests of this study.

DISCLAIMER
This in-vitro study initiative has been funded by Johnson and Johnson (Philippines), Incorporated through a research grant to Asian Hospital, Inc.