Effectiveness of Preprocedural Mouthwashes: A Triple-Blind Randomised Controlled Clinical Trial

doi:10.21203/rs.3.rs-4018100/v1

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Effectiveness of Preprocedural Mouthwashes: A Triple-Blind Randomised Controlled Clinical Trial

https://doi.org/10.21203/rs.3.rs-4018100/v1

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Objective: to determine and compare the effectiveness of three Government-recommended pre-procedural mouthwashes in the mitigation of respiratory pathogens during dental care.

Material and Methods: 228 participants were block-randomised to the pre-procedural mouthwash groups: Povidone-iodine, Hydrogen Peroxide, and Chlorhexidine Digluconate. Participants rinsed with mouthwashes prior to dental treatment. Participants, operators, and assessors were blind to the assigned mouthwashes (triple blind). A multitude of respiratory pathogens (19 viruses including SARS-CoV-2) were assessed. Changes in the prevalence and mean number of ‘any’ pathogen were determined.

Results: All completed the trial and there were no significant differences in the demographic profile with respect to intervention groups (all, P>0.05). The prevalence of ‘any’ detected respiratory viral pathogens in the pre-procedural saliva was 3.5% (8/228) compared to 1.3% (3/228) in post-procedural saliva, P=0.034. The mean number of viruses reduced significantly following pre-procedural mouthwash use: 0.04 (SD 0.18) to 0.01 (SD 0.11), P=0.025. There was no significant difference in the reduction (∆) of ‘any’ detected virus (prevalence), P=0.155, nor any significant reduction in mean number (∆) of ‘any’ detected virus in the post-procedural saliva compared to the pre-procedural saliva of participants with respect to mouthwash used, P=0.375.

Conclusions: The HKSAR recommended practice of pre-procedural mouthwash use was effective in the mitigation of respiratory pathogens during dental aerosol-generating treatment.

Clinical Relevance: The present study lends support for the HKSAR’s policy on pre-proceduralmouthwashes in the mitigation of respiratory pathogens during dental care, this has implications for practice and policy during pandemics.

Pre-procedural mouthwash

dentistry

respiratory pathogens

SARS-CoV-2

Respiratory pathogens can give rise to a range of infections including pneumonia, which has long been considered a worldwide public health issue [1, 2]. The global burden of respiratory infections in terms of mortality and morbidity is considerable both for adults and children, and particularly in low- and middle-income countries [3]. The mouth can serve as a portal entry for respiratory pathogens and a site where they can replicate at an alarming rate, particularly among those medically compromised and when oral hygiene is neglected [4]. In addition, dental prosthesis (such as dentures), as non-shedding surfaces, can harbor respiratory pathogens when prosthesis are not removed at night-time and/or when there is inadequate denture care [5]. Respiratory pathogens have been frequently detected from oral mucosae (buccal swab samples), oral fluids (saliva and gingival crevicular fluid) and denture surfaces, highlighting the risk for self-infection and transmission to others [4, 6]. Sampling the mouth offers a non-invasive, easily accessible way of detecting the presence of respiratory pathogens and respiratory diseases, with saliva being widely used in diagnosis [6].

A key concern is that respiratory pathogens in the mouth can be easily aspirated leading to a range of infections, including acute respiratory infections and ‘aspirational pneumonia’ [7]. Analyses of respiratory sputum samples have confirmed that based on microbial composition with evidence of specific oral pathogens and salivary contaminants, that the mouth can act as a common source of and reservoir for respiratory infection [8]. This of particular concern among frail elders and immunocompromised individuals as it associated with considerable morbidity and mortality [9]. To this end oral hygiene care is key and important to maintain in critically ill patients to prevent ventilator-associated pneumonia [10].

The mouth can also act as a key site for transmission of respiratory pathogens. Aerosols from speaking, coughing/sneezing are widely perceived as a dominant path for airborne transmission for those in close contact with infected individuals [11]. Moreover, such bioaerosols can lead to contamination of the immediate environments, both air and surfaces [12]. Furthermore, contact with oral tissues including saliva can lead to rapid transmissions of respiratory pathogens [13]. In addition, many dental procedures are aerosol generating such as with the use of ultrasonic scalers for tooth cleaning (scaling) and powered handpiece for tooth preparation (drilling) and this leads to aerosols of a wider reach/range and the production of droplets that can exacerbate the spread of larger respiratory infectious particles [12, 14, 15].

COVID-19 as a global public health crisis lead to widespread illness, death and economic disruptions of pandemic proportions [16]. The transmission of SARS-CoV-2 by aerosol was the dominant mode for contracting COVID-19 infection and thus there was increased awareness of the mouth as a reservoir of infection [11]. The wearing of facemasks was a key public health measures to control COVID-19 infection and protect against transmission of SARS-CoV-2 between individuals in the community [17]. There was particular concern about the risk of transmission of SARS-CoV-2 in the dental setting given the close proximity of oral health care workers to patients, direct contact / exposure to the oral environment and because of dental aerosol generating procedures [18]. The World Health Organization advised to limit dentistry to ‘essential dental care’ given the potential high risk of transmission [19]. As oral health is an integral aspect of health, various national and international organizations produced guidelines and provided recommendation to mitigate against respiratory pathogens by personal protective equipment, judicious use of aerosol generating equipment and other measures. The use of preprocedural mouthrinses was widely advocated among guidelines to obviate bio-aerosols: recommend by more than 80% of guidelines [20, 21].

The use of preprocedural mouthwashes has long been suggested to reduce microbial load before dental and oral surgery procedures to prevent oral contamination (by way of spatters and aerosols) and reduce post-operative complications [22]. With the advent of COVID-19 there was an increase in the use of preprocedural mouthwashes and this has remained in line with local guidelines [23, 24]. However, there is limited and conflicting evidence as to the effectiveness of preprocedural mouthwashes in mitigating against COVID-19 and other respiratory infections [25]. Given the paucity of information, this study aimed to determine and compare the effectiveness of preprocedural mouthwashes recommended by the Department of Health of the Hong Kong Special Administrative Region (HKSAR) to support or refute their continued used in dental practice [26].

Study Design, Participants & Setting

This study was a parallel-arm, triple-blind, randomized controlled trial. Ethical approval for the trial was obtained from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (#UW 21-129) and the clinical trial was registered with the Chinese Clinical Trial Register (ChiCTR2200064784). Participants were recruited from Philip Prince Dental Hospital and University Health Service Clinics by way of telephone interview and a brief clinical screening was performed for suitability to participate at the Institute for Advanced Dentistry Clinical Research Centre, HKU Dentistry. The inclusion criteria were (i) be aged 18 or above; (ii) no known allergy to mouthwashes; (iii) not taking or have taken any anti-viral, anti-bacterial or anti-fungal agents-topically or systemically in the past month; (iv) no contraindications to use of mouthwashes such as thyroid disease, or undergoing radioactive iodine therapy (which may interact with povidone -iodine).

Interventions

The three intervention agents were the Government recommended mouthwashes for use in the mitigation of COVID-19 and respiratory infections [26]: (i) Povidone-iodine 1% w/v, Betadine® (PVP-I), with instruction to rinse with 10mL for 30s and then spit out; (ii) Hydrogen Peroxide 1.5% w/v, Peroxyl®-Colgate® (H₂O₂), with instruction to rinse with 10mL for 1 min and then spit out; (iii) Chlorhexidine Digluconate 0.2% w/v, Corsodyl® (CHX), with instruction to rinse with 10 mL for 1 min and then spit out. The mouthwashes were administered by the attending nurse to patients at the Clinical Research Centre approximately 15 min prior to the assigned dental prophylaxis treatment (scale & polish).

Samples Collection & Outcome Assessments

Prior to administration of the intervention mouthwashes, unstimulated saliva samples were collected [Pre-Pre-Procedural Saliva Mouthwash Sample]. Participants refrained from eating, drinking or smoking for at least 30 minutes prior to sample collection. Whole unstimulated saliva samples were collected using the drooling method [27]. Standardised instructions were given to the patients to lean slightly forward on the dental chair and allow for saliva to pool in their mouth and not to swallow. Then to allow the saliva to drool into a 15 mL sterile centrifuge tube (Corning, USA). The time set for saliva collection was approximately 5 minutes to obtain 1-2 mL of saliva. The saliva samples were sealed and labeled and temporarily stored on ice in preparation for downstream analyses. This procedure was replicated following the administration of the assigned mouthwash [Post-Pre-Procedural Saliva Mouthwash Sample]. The harvested samples were dissolved in a viral transport medium (Working Earle’s BSS, 5%BSA, 20% glucose, working vancomycin, working Amikacin, working Nystatin and 4.4% NaHCO₃) to maintain the viability and integrity of samples [28] in 5 mL Axygen transport tubes (Corning, USA) and they were stored in -80 ℃ (Haier Biomedical, China).

Environmental samples were collected in two ways (i) air samples and (ii) working surface samples. Air Samples were collected using an MD8 Airport Portable Air Sampler with standardised Gelatine Membrane Filters (Sartorius Scientific Instruments, China). The air sampler for the monitored area was set with a flow of 50 L/min and a total volume sampled of 2 m³. The air sampler was positioned at approximately 1.5 m above the floor and 1m horizontal distance from the head rest of the dental chair where the dental scaling was carried out, Figure 1. At the beginning of each clinical session prior to admittance of patients’ air samples were obtained as control air samples. The membrane from the filters was sectioned into small pieces using a sterile forceps and immersed into 2 mL viral transport medium in 5 mL Axygen transport tube, sealed and then were stored in -80 ℃.

Working surface samples of the dental chair tray and instrument holder were obtained by swabbing surfaces with a disposable specimen collection flocked swabs (Medico, China) on completion of dental treatment, Figure 1. Control samples were obtained from the dental chair tray and instrument holder prior to any dental treatment. The swabs were immersed in 2 mL viral transport medium in 5 mL Axygen transport tube, sealed and then were stored in -80 ℃.

Outcomes

The primary outcome is the reduction in the prevalence of respiratory pathogens as identified from the NxTAG^® Respiratory Pathogen Panel + SARS-CoV-2 test (Diasorin, Italy). Assessment of the presence of ‘any’ respiratory pathogens is based on qualitative simultaneous detection of nucleic acids from multiple respiratory viruses in saliva samples. The secondary outcomes include the reduction in the prevalence of respiratory pathogens determined by air samples and swabs samples. The NxTAG^® respiratory panel detects 19 viral pathogens including SARS-CoV-2: Adenovirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Human Bocavirus, Human Metapneumovirus, Influenza A, Influenza A – subtype H1, Influenza A – subtype H3, Influenza B, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, Rhinovirus/Enterovirus, SARS‑CoV‑2 (ORF1ab and M gene).

For the extraction of total nucleic acid, 10μL of MS-2 internal control was added to monitor the extraction and amplification steps, together with 200 μL of either saliva or environmental sample and mixed using Tianlong’s viral RNA and DNA extraction kit (T016H, Tianlong Science & Technology, China) in accordance with the manufacturer’s instructions. 60 μL elution volume of total nucleic acid (TNA) for each sample was acquired. For further testing, 35 μL pure extracted TNA was spiked into pre-plated NxTAG^® Respiratory Pathogen Panel wells containing lypholized reagents and re-suspended gently on ice. Subsequently, the RT-PCR amplification was performed using an Applied Biosystems (Thermo Fisher Scientific, USA), with the detailed cycling parameters as one reverse transcription step at 42°C for 20 min; one template denaturation step at 95°C for 2 min and 30 s; 15 first amplification cycles at 95°C for 20 s, 65°C for 60 s, and 72°C for 10 s; 24 nested amplification cycles at 95°C for 20 s, 58°C for 60 s, and 72°C for 10 s; and a hybridization step at 37°C for 45 min. Subsequently, the plate was transferred to the 37°C preheated MagPix heater plate of the MagPix instrument (Luminex Corporation, Austin, TX). SARS-CoV-2 TNA and MS-2 were used for each single batch as positive and negative control, respectively. The total turnaround time was around 4h. The results were analyzed via Luminex SYNCT™ software 1.1. (Luminex Molecular Diagnostics, Toronto, Canada), Figure 2.

Sample size

In this study, the primary outcome is the reduction in the prevalence of respiratory pathogens by administering pre-procedural mouth rinsing. A previous study conducted in the University of Hong Kong identified that the prevalence of ‘any’ Respiratory Pathogen from saliva, as detected by NxTAG^® Respiratory Pathogen Panel IVD was 23.4% among those with non-fever/ non-respiratory symptoms attending [29]. In determining the sample size/ sample power for this clinical trial the assumption was that patients attending the dental clinical would have a similar prevalence of respiratory pathogen (i.e. non-fever/ non-respiratory symptom patients 23.4%) and that ‘willingness to participate’ rate would be ~85% (as identified in a pilot study). Then in order to detect a significant change in the prevalence of ‘any’ respiratory pathogen following mouth rinsing a sample size of 196 would have 80% statistical power, using McNemar test (paired categorical data). To allow for ~15% drop-out it seemed prudent to recruit 228 participants.

Randomization of Participants

Randomization and allocation sequence were computer generated by an officer of the Translation Research Laboratory of the faculty who was not involved in recruitment or assessment of study participants and based on random digit generation with participants in blocks of three. Subject allocation was concealed in sequentially numbered opaque sealed envelopes (SNOSE) by an administrative assistant not involved with the study [30]. The attending clinical trial nurse opened envelopes after registration of the patient at clinical trial centre and provided the intervention (pre-procedural mouth rinse) in a room adjoining the dental surgery. In the present study, the participants were blinded to what mouth rinse they received as the mouth rinse were provided in a similar dispensing cup of similar volume (10 mL). The operators in the dental clinic and the research assistant collecting samples were blinded to what pre-procedural mouth rinse the participants had received as they were not present when mouthwashes were administered.

Statistical Analyses

Firstly, the data output from NxTAG^® Respiratory Pathogen Panel was processed by the Luminex SYNCT™ software 1.1. (Luminex Molecular Diagnostics, Toronto, Canada) for (i) pre-procedural saliva samples, ii) post-procedural saliva samples, iii) environment control air samples (air samples prior to dental treatment), iv) environment air samples (post-treatment), v) control environment swabbed work surface (pre- dental treatment) and vi) environment swabbed work surface following treatment. The data on detection of the 19 viruses (detected (1), not detected (0)) was entered for saliva samples (pre and post), air samples (pre (control) and post) and swabbed-surfaces (pre (control) and post) into SPSS 27.0 IMB® SPSS® Statistics, USA). In addition, demographic details of participants were entered: age (continuous variable), gender (nominal/ binary categorical) and educational attainment level (ordinal categorical).

The demographic profile of participations among the three intervention groups were compared (employing Chi-Square tests for categorical variables and One-Way-Analyses of Variance (ANOVA) for age (continuous variable). Descriptive statistics were produced of the prevalence of each of the 19 type of virus (number, %), prevalence of ‘any’ detected viruses (number, %), and the number of viruses (range, mean (standard deviation)). The prevalence of ‘any’ detected virus in pre-procedural saliva and post-procedural via was compared using the McNemar test for paired categorical data comparisons. Change in the prevalence (∆) of ‘any’ detected virus in the pre-procedural saliva to the post-procedural saliva of participants was determined, and variations in the change in the prevalence (∆) between the three intervention groups was compared using Chi-Square tests. Following on, the mean number of detected virus in the pre-procedural saliva was compared to mean number of detected virus in the post-procedural saliva using the Wilcoxon signed-rank test (non-parametric equivalence of paired-t test). Changes in the number (∆) of ‘any’ detected virus in the pre-procedural saliva to the post-procedural saliva of participants was determined, and variations in the change in the number (∆) between the three intervention groups was compared using Kruskal Wallis tests (non-parametric equivalence to ANOVA).

All environmental post-treatment swabbed surfaces were analysed for presence of (prevalence) of each of the 19 types of viruses (number, %), prevalence of ‘any’ detected viruses (number, %), and the number of viruses (range, mean (standard deviation). Comparisons to pre-treatment swabbed surfaces to be made if applicable (virus detected in post-treatment swabbed samples) using statistics as outline above. A random sample of environmental air samples (post-treatment) were analyzed for presence (prevalence) of each of the 19 types of viruses (number, %), prevalence of ‘any’ detected viruses (number, %), and the number of viruses (range, mean (standard deviation)). Likewise, comparisons to control pre-treatment environment air samples was made if applicable (virus detected in post-treatment environment samples) using statistics as outline above.

Participation and Profile of Participants.

Recruitment and screening of participants took approximately four months. Following clinical screening, 228 out of 380 subjects were suitable and amenable to participate in the clinical trial. The 228 participants were randomly allocated through block randomization (in blocks of three) to three intervention groups (CHX, PVP-I and H₂O₂); 76 participants per group. Each participant was given a 1-hour appointment time for dental treatment at the clinical trial centre. A flow diagram of the clinical trial process is presented in Figure 3. The age of participants ranged from 19 to 86, with a mean age of 48.3 (18.5), 50.4% (115) were male, and 44.3% (101) had attained university/ college level education. There was no significant difference in the profile of participants with respect to age (P>0.05), gender (P>0.05) nor educational attainment level (P>0.05), Table 1.

The prevalence of ‘any’ detected respiratory viral pathogens in the pre-procedural saliva was 3.5% (8/228). Among eight individuals five viruses were detected: Respiratory Syncytial Virus A (1, 0.44%), Parainfluenza 4 (1, 0.44%), Influenza B (1, 0.44%), Rhinovirus/Enterovirus (3, 1.32%), SARS-CoV-2 (ORF1ab) (2, 0.88%) SARS-CoV-2 (M) (2, 0.88%). No individual had more than one virus detected. Overall, the mean number of viruses detected was 0.04 (SD 0.18).

Following the preprocedural mouthwash use, the prevalence of subjects with any viruses detected in their post-procedural saliva was 1.3% (3/228). Among the three individuals two viruses were detected: Rhinovirus/Enterovirus (1, <0.01%) and SARS-CoV-2 (ORF1ab) (2, 0.88%) and SARS-CoV-2 (M) (2, 0.88%). No individual had more than one virus detected. Overall, the mean number of viruses detected was 0.01 (SD 0.11).

There were significant changes in the prevalence of any respiratory pathogen from saliva samples pre and post mouthwash use: 3.5% (3/228) to 1.3% (3/228), p=0.034, Figure 4. Following preprocedural mouthwash use Respiratory Syncytial Virus A, Parainfluenza and Influenza B were not detected. In two of the three samples where Rhinovirus/Enterovirus was detected in the saliva pre-procedural mouthwash use it was no longer detected in the post-procedural mouthwash saliva sample. The SARS-CoV-2 remained detectable as assessed by SARS-CoV-2 (ORF1ab) and SARS-CoV-2 (M) in post-procedural mouthwash saliva as was detectable in the saliva sample pre-mouthwash use. The mean number of viruses detected reduced significantly following preprocedural mouthwash use: 0.04 (SD 0.18) to 0.01 (SD 0.11), p=0.025.

There was no significant change in the reduction of the prevalence (∆) of ‘any’ detected virus in the pre-procedural saliva compared to the post-procedural saliva of participants with respect to preprocedural mouthwash used, p=0.155. Among the PVP-I group 1 participant had a detectable respiratory virus (SARS-CoV-2 as detected by SARS-CoV-2 (ORF1ab) and SARS-CoV-2 (M)) in their saliva pre-procedural mouthwash use and it was also detectable in saliva post mouthwash use. Among the CHXgroup, 4 participants had a detectable respiratory virus in their saliva pre-mouthwash use: Respiratory Syncytial Virus A (1 participant), Rhinovirus/Enterovirus (2 participants), and 1 participant with SARS-CoV-2 (detectable by SARS-CoV-2 (ORF1ab) and SARS-CoV-2(M). Following CHX mouth use, 2 participants had a detectable respiratory virus in their saliva (Rhinovirus/Enterovirus (1 participants) and 1 participant with SARS-CoV-2 (detectable by SARS-CoV-2 (ORF1ab) and SARS-CoV-2(M). Among the PI group 1 participant had a detectable respiratory virus (SARS-CoV-2 as detected by SARS-CoV-2 (ORF1ab) and SARS-CoV-2 (M)) in their saliva pre-procedural mouth wash use and it was also detectable in saliva post mouthwash use. Among the H₂O₂group, 3 participants had a detectable respiratory virus (Parainfluenza 4, Influenza B, and Rhinovirus/Enterovirus) and no virus was detected in the saliva post mouthwash use.

There was no significant reduction in the mean number (∆) of ‘any’ detected virus in the post-procedural saliva compared to the pre-procedural saliva of participants with respect to preprocedural mouthwash used, p=0.341. Among the CHXgroup, the mean number of viruses detected in the post-procedural saliva compared to pre-procedural mouthwash reduced: 0.05 (SD 0.22) versus 0.03 (SD 0.16). Among the PVP-Igroup, the mean number of viruses detected in the post-procedural saliva compared to pre-procedural mouthwash was similar: 0.01 (SD 0.11) – the case of SARS-CoV-2 remained. Among the H₂O₂group, the mean number of viruses detected in the post-procedural saliva compared to pre-procedural mouthwash reduced: 0.04 (SD 0.20) versus 0.00 (SD 0.00).

An analysis of air samples failed to identify any respiratory pathogen among the NxTAG^® Respiratory Pathogen Panel. Likewise, among the surface-swabbed surfaces post-dental treatment no respiratory pathogen was detected from the among the NxTAG^® Respiratory Pathogen Panel.

Transmission of infections in the dental setting has long been of concern, and particularly airborne pathogens that can be spread in the surgery through aerosol general procedures [15]. Thus, perhaps not surprisingly there were concerns and limitations to dental practice during the COVID-19 pandemic [31]. Personal protective equipment (PPE) are the mainstay practices implemented for universal infection control, and with upgrades in type of PPE, such as filtering facepiece respirators, greater protection against airborne pathogens was achieved [32]. As an adjunct measure the practice of preprocedural mouthwashes was advocated, and was a recommendation adapted by the Department of Health and implemented at all government clinics and hospitals; and remains so in 2024 [26]. This clinical trial aimed to address whether the practice of preprocedural mouthwash use was effective against respiratory pathogens (including SARS-CoV-2), and to compare the effectiveness of the three recommended mouthwashes CHX, PVP-I and H₂O₂to support or refute the practice.

The study benefited from being a triple blind randomised controlled trial and thereby limited potential bias in terms of patient, clinician, and researcher with respect to mouthwash use, aerosols generated, collection of samples, and laboratory analyses [33]. Given that the practice of preprocedural mouthwash was implemented at part of usual care (as per Government recommendation) it was feasible to recruit participants for the trial within a short period of time. To allow for fallow time with adequate ventilation and cleaning a limited number of appointments were allocated per day, but it was still feasible to complete the trial in approximately three-months [34].

Among participants, approximately one-in-thirty had a detectable respiratory pathogen in their saliva pre-dental treatment (pre-procedural mouthrinse). Only two participants had SARS-CoV-2 detected in their pre-treatment saliva as detectable by SARS-CoV-2 (ORF1ab) that detects specific genetic sequences of the virus from open reading frame and detected by SARS-CoV-2(M) membrane protein [35]. The low prevalence of detectable respiratory pathogens including SARS-CoV-2 maybe attributed to the range of practices and measures implemented in the community and the hospital during the period of the clinical trial. At the community level strict quarantine measure at designated centers was in place for travelers to Hong Kong, widespread testing to identify and isolate SARS-CoV-2 infected individuals, and extensive contact tracing to identify and alert those who may have been exposed to the virus following Hong Kong’s 5^th wave of Covid-19 [36]. Mandatory mask wearing requirements was continued until March 2023 and there were reports that because of such practice the prevalence of respiratory infections (detection of influenza and coronavirus) was low in the community [37]. At the hospital level, temperature screening, COVID-19 symptom checklist declaration and vaccination was required for elective dental treatments at dental hospital, in line with practices elsewhere.

There was a significant reduction in the prevalence of ‘any’ detected respiratory viral pathogens in the saliva after the pre-procedural mouthwash use compared to before the pre-procedural mouthwash use, with elimination of Respiratory Syncytial Virus (CHX use), Parainfluenza 4 (H₂O₂use) and Influenza B (H₂O₂use). Among two of the three participants Rhinovirus/Enterovirus was eliminated, it was eliminated in the one case after H₂O₂use, and it was eliminated in one of the two cases after CHX use. Several in vitro studies have found mouthwashes to been effective against respiratory pathogens: CHX (0.12%) has virucidal effectiveness against influenza A and parainfluenza [38]; PVP-I (0.23%) mouthwash has virucidal effectiveness against Influenza virus A, SARS-CoV, and MERS-CoV [39].

However, among the two individuals with detectable levels of SARS-CoV-2 in their saliva before the pre-procedural mouthwash use, SARS-CoV-2 remained detectable after the mouthwash use, after use of CHX (1 case) and after use of PVI (1 case). In our clinical trial the outcome measure was detectable presence of SARS-CoV-2 as assessed NxTAG^® Respiratory Pathogen Panel as determined by multiplexed polymerase chain reaction [40]. In contrast, others have focused on SARS-CoV-2 viral load using qRT-PCR and based on cyclic threshold levels (Ct) [41] or cytopathic effects of SARS-CoV-2 as determined by 50% tissue culture infectious dose assay (TCID50) as a marker of infectivitiy [42]. However, a high level of agreement in dectection of SARS-CoV-2 from NxTAG^® Respiratory Pathogen Panel and routine COVID-19 qRT-PCR protocol assesment methods was reported: Kappa 0.98 [40]. There has been conflicting evidence as to the effectiveness of pre-procedural mouthwashes on SARS-CoV-2, and this in part relates also to differences in protocols of pre-procedural mouthwash use – type of active agent, concentration, volume, and period of gargling. For example, Seneviratne et al. [43] did not find rinsing with CHX 0.12%, 15 mL for 1-minute effective at reducing SARS-CoV-2 Ct levels wheras Chaudhary et al. [44] and Costas et al. [41] did identify CHX (0.12%, 15 mL, 1-minute) to be effective at reducting CoV-2 Ct levels. Bonn et al. [42]reproted that a CHX contaning mouthwash (with CPC) was effective at reducing SARS-CoV-2 Ct and SARS-CoV-2 TCID50 levels. Similarly, there was conflicting evideince as to the effectiveness of PVI-P: Ferrer et al. [45] did not find rinsing with PVP-I 2.0%, 15 mL for 1-minute effective at reducing SARS-CoV-2 Ct levels in saliva, wheras Seneviratne et al. [43] did find PVP-I 0.5%., 10 mL for 30s to be effective at reducing SARS-CoV-2 Ct levels despite a lower concentration of PVP-I being used and gargeled for half the time. Chaughary et al. [44] also found rinsing with PVP-I 0.5%., 15 mL for 1 minute to be effective at reducing SARS-CoV-2 Ct levels despite a lower concentration. In our clinical trial no particiapant assigned to the H₂O₂group had SARS-CoV-2 detected in their saliva so as to report on its effectiveness against it. However, Chaughary et al. [44] found rinsing with H₂O_2,1.0%., 15 mL for 1 minute to be effective at reducing SARS-CoV-2 Ct level and other mouthwashes have also been reported to be effective against SARS-CoV-2, for example, cetlypyridinium chloride- CPC (0.075%, 20 mL, 30s) in terms of SARS-CoV-2levels Ct levels [43] but Ferrer et al [45] did not find CPC to be effective against SARS-CoV-2.

No viral pathogens were detected in environmental samples – both swabbed-surface samples and air samples (post dental treatment). In hospital clinic environments swabbed-surface samples and air samples have detected respiratory pathogens including SARS-CoV-2 [46]. There have been concerns of potential transmission of respiratory pathogens in the dental office from aerosols, droplets and contamination of working surfaces [15, 47]. Moreover, in the time of COVID-19 pandemic there were particular concerns about SARS-CoV-2 transmission through aerosol generating procedures (AGP), so much so that AGPs were restricted in certain settings [15]. The detection of no viral pathogens in the environmental samples during our clinical trials despite AGPs been used could be attributed to the infection control measures and high-volume suction in line with other findings [48].

In summary, the findings of this triple-blind randomised controlled clinical trial found pre-procedural mouthwashes to be effective in reducing the prevalence and number of detectable respiratory pathogens overall, but not SARS-CoV-2 specifically. These findings should be interpreted in light of the limited number of participants with ‘any’ detectable respiratory pathogens, and the limited number and type of respiratory pathogens detected. There was no significant difference in the effectiveness of the three-government recommended pre-procedural mouthwashes, suggesting that any of the three HKSAR government recommend pre-procedural mouthwash may be used in clinical practice.

Author Contributions

All authors contributed to the study conception and design. Conceptualization, funding acquisition, supervision, methodology, formal analysis & writing – review and editing by [McGrath Colman]. Writing – review and editing by [Leung Yiu Yan]. Conceptualization, methodology & writing – review and editing by [Neelakantan Prasanna]. Supervision & writing – review and editing by [Chan Kwok Hung]. Methodology & writing – review and editing by [Leung Joy Ka Yi]. Resources, supervision, methodology & writing – review and editing by [Hung Fan Ngai]. Material preparation, data collection, formal analysis & writing – original draft by [Huang Shan]. All authors commented on previous versions of the manuscript and read and approved the final manuscript.

Ethics Approval and Consent to Participate

Ethical approval for the clinical trial was obtained from the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (#UW 21-129). Written informed consents were obtained from all participants.

Clinical Trial Registration

This triple-blind randomized clinical trial was registered at www.chictr.org.cn with the registry number (ChiCTR2200064784).

Funding

This clinical trial was supported by the General Research Fund of the Research Grants Council of the Hong Kong Special Administrative Region, China (Grant number: 17107621).

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

Sincere appreciation to Ms. Cindy P.W. Yeung for the help of enrollment of the participants, negotiation during the clinical trial and data storage. Special thanks to Ms. Pauline Ho Po Ling for her communication with the local participants and intervention administration during the clinical trial.

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Table 1. Demographic Profile of Participants among Three Intervention Groups

		Group I-PVP-I (n=76)	Group II-H₂O₂ (n=76)	Group III-CHX (n=76)	Total (n=228)	p
		n (%)				p
Age (years)	18-44	34（44.7）	36（47.4）	32（42.1）	102(44.7)	0.892
	45-69	19（25.0）	11（14.5）	13（17.1）	43(18.9)
	70-	23（30.3）	29（38.2）	31（40.8）	83(36.4)
Gender	Female	34（44.7）	38（50.0）	41（53.9）	113(49.6)	0.522
Gender	Male	42（55.3）	38（50.0）	35（46.1）	115(50.4)	0.522
Educational Attainment	University/College	34（44.7）	35（46.1）	32（42.1）	101(44.3)	0.944
	Secondary school	35（46.1）	33（43.4）	34（44.7）	102(44.7)
	Primary/ Below	7（9.2）	8（10.5）	10（13.2）	25(10.9)

Table 2. The Prevalence of Respiratory Viruses Found in Saliva: Pre and Post

	Pre-Mouthwash Saliva	Percentage (%)	Post-Mouthwash Saliva	Percentage (%)
Any respiratory virus	8	3.51	3	1.32
Respiratory Syncytial Virus A	1	0.44	0	0
Parainfluenza 4	1	0.44	0	0
Influenza B	1	0.44	0	0
Rhinovirus/Enterovirus	3	1.32	1	0.44
SARS-CoV-2 (ORF1ab) SARS-CoV-2 (M)	2	0.88	2	0.88
SARS-CoV-2 (ORF1ab) SARS-CoV-2 (M)	2	0.88	2	0.88

No competing interests reported.

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Effectiveness of Preprocedural Mouthwashes: A Triple-Blind Randomised Controlled Clinical Trial

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