key: cord-0864608-4943fibq authors: ElZein, R.; Abdel Sater, f.; Fakhreddine, S.; Abi Hanna, P.; Feghali, R.; Hamad, H.; Ayoub, F. title: In vivo evaluation of the virucidal efficacy of Chlorhexidine and Povidone-iodine mouthwashes against salivary SARS-CoV-2. date: 2021-03-17 journal: nan DOI: 10.1101/2021.03.07.21252302 sha: c5ceb6b6746ca341571ea03eb13b1d0b013c9311 doc_id: 864608 cord_uid: 4943fibq Abstract Background. The oral cavity is potentially high-risk transmitter of COVID-19. Antimicrobial mouthrinses are used in many clinical pre procedural situations for prophylactic purposes. An evident-based investigation for an effective mouthwash solution against salivary SARS-CoV-2 is urgently required for the exposure reduction during dental procedures. Aims. This study aimed to evaluate in vivo virucidal efficacy of 2 mouthwashes: 1% Povidone-iodine and 0.2% Chlorhexidine as a dental preprocedural oral disinfection against salivary SARS-CoV-2. Materials and Methods. In this randomized-controlled clinical trial, studied group comprised laboratory-confirmed COVID-19 positive patients through nasopharyngeal swabs. Participants were divided into 3 groups. For 30 seconds, group A gargled with 1% Povidone-iodine, group B mouthrinsed with 0.2% Chlorhexidine and control group C mouthrinsed with distilled water. Saliva samples were collected before and 5 minutes after mouthwash. SARS-CoV-2 rRT-PCR was then performed for each sample. Evaluation of the efficacy was based on difference in Ct value. The analysis of data was carried out using GraphPad Prism version 5 for Windows. Paired t test and unpaired t test were used. A probability value of less than 0.05 was regarded as statistically significant. Results. Sixty-one compliant participants (36 female and 25 male) with a mean age 45.3 plusmn 16.7 years-old were enrolled. A significant mean Ct value difference (p < 0.0001) between the paired samples in group A (n = 25) and also in group B (n = 27) (p < 0.0001) was found. In contrast, no significant difference (p = 0.566) existed before and after the experiment in the control group C (n = 9). Moreover, a significant difference was noted between the delta Ct of distilled water wash and each of the 2 solutions 1 % Povidone-iodine (p = 0.012) and Chlorhexidine 0.2% (p = 0.0024). No significant difference was found between the delta Ct of patients using 1% Povidone-iodine and Chlorhexidine 0.2% solutions (p = 0.24). Conclusion. Chlorhexidine 0.2% and 1% Povidone-iodine oral solutions are effective preprocedural mouthwashes against salivary SARS-COV-2 in dental treatments. Their use as a preventive strategy to reduce the spread of COVID-19 during dental practice should be systematically implemented. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel member of the human coronaviruses from the Coronaviridae family and belongs to the Betacoronavirus genus. 1 The subsequent corona virus disease-2019 (COVID-19), rapidly spreading worldwide, 2 mostly causes respiratory disorders. 3, 4 The human-to-human transmission of SARS-CoV-2 essentially happens by inhalation of respiratory droplets spread by coughing or sneezing from an infected person, and by direct contact of contaminated surfaces followed by touching the nose, mouth and eyes. 5, 6 The virus can even survive on various surfaces for days. 7 Transmission via ocular has been shown. 8 The oral cavity is potentially high-risk transmitter of COVID-19. In fact, when operating dental treatments with the high-speed handpiece, it is fundamental to use a water coolant, 9 generating consequently aerosols mixed with saliva or blood. These bioaerosols, generally contaminated with microorganisms including bacteria, fungi, and viruses, float in the air then settle on the surfaces and can be transmitted to the dentists or other patients by inhalation or contact. 10 ,11 SARS-CoV-2, was identified in saliva of infected patients. 12 Furthermore, it has been reported that the main cell receptor of SARS-CoV-2, angiotensinconverting enzyme II (ACE2), is extremely expressed on the mucosa of the oral cavity and particularly in the epithelial cells of the tongue. 13 Therefore, it is crucial for dental practitioners to install preventive strategies to avoid the COVID-19 infection by focusing not only on patient placement, hand hygiene, all personal protective equipment, caution in performing aerosolgenerating procedures but also on patient's pre-procedural antiseptic mouthrinse. 14, 15 In fact, antimicrobial mouthrinses are an important part of oral care. Such solutions are used in many clinical pre procedural situations for prophylactic purposes. 16, 17 Preprocedural oral solution is one of the most effective methods of reducing the amount of microorganisms in oral aerosols. 18, 19 In addition, gargling is also assumed to produce favorable effects through removal of oral and pharyngeal protease that helps viral replication . 20 Thus, an investigation for an effective mouthrinse against COVID-19 is urgently required for the control of oral and respiratory tract infection and for the exposure reduction during dental procedures. In the literature, it was reported that a pre-procedural 0.12% Chlorhexidine mouth rinse can reduce the microbial load of saliva. 21 A meta-analysis showed that the use of preprocedural mouth rinse, including Chlorhexidine, essential oils, and cetylpyridinium chloride, resulted in a mean reduction of 68.4% colony-forming units in dental aerosol. 22 Although the effect of Chlorhexidine gluconate on human coronavirus is unknown but it is effective against many respiratory viruses, like herpes and HIV. 23 On the other hand, Povidone-iodine is a broad-spectrum antimicrobial that has been used in infection control and prevention for over 60 years and is available in various preparations for use as a disinfectant for the skin, hands and mucosal surfaces, as well as for wound treatment and eye applications. 24 Povidone-iodine has well-established general antimicrobial activity, demonstrating in vitro efficacy against wide range of enveloped and nonenveloped viruses. [25] [26] [27] Recent in vitro study has demonstrated rapid virucidal its products activity against MERS-CoV. 28, 29 The benefit of gargling with Povidone-iodine has also already been noted in Japanese clinical respiratory guidelines. 30 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 17, 2021. ; This study, besides of being an additional research concerning the Consistency of detection of SARS-CoV-2 in saliva from the Lebanese experience, aimed mainly to evaluate in vivo virucidal efficacy of 2 mouthrinses: 1% Povidone-iodine and 0.2% Chlorhexidine as a dental preprocedural oral disinfection against salivary SARS-CoV-2. Ethical approval. Ethical clear of this research was delivered from the Lebanese University Institutional Review Board (#CUER 13-2020). The study was conducted in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All participants were informed about the study and gave their consent. It was conducted between June and Septembre 2020 at the isolation ward of Rafik Hariri University Hospital (RHUH) of Beirut for sampling and in the Laboratory of Cancer Biology and Cellular Immunology, COVID-19 Unit, Faculty of Sciences, Lebanese University for viral PCR tests. Trial design. This study was a parallel group, quadruple blind-randomized-placebo-controlled clinical trial with an add on laboratory based study. Patients, intervention supervisor, laboratory technicians and the data collection subject were blinded. The groups were labelled as A, B and C. The codes of the intervention were only revealed at the end of study. A non-probability, purposive sampling technique was adopted. Studied group comprised, laboratory-confirmed COVID-19 positive patients, through nasopharyngeal swabs. The lapse of time between COVID-19 diagnosis and inclusion in the study ranged from zero to two days. Were excluded cases indicated for intubation or mechanical ventilation and patients who declined consent. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 17, 2021. ; Solution preparation. 15 ml of each undiluted mouthwash solution were previously poured into a sterile cup within a Biosafety cabinet (Topair system) in the Laboratory of Cancer Biology and Cellular Immunology, Faculty of Sciences, Lebanese University with respect to the manufacturer recommendations for each solution. The containers were marked as A, B or C, and then delivered to RHUH to accomplish the sampling. Solution A referred to 1% Povidone-iodine, B for 0.2% Chlorhexidine and C for distilled water as a placebo treatment. Sampling. Participants were randomly divided into the three groups. The same trained operator explained, provided and supervised the sampling in patient's room with respect to COVID-19 infection control. Sampling was performed by the patients themselves in the early morning on empty stomach and before brushing teeth. First, participants were asked to cough out saliva from throat (2ml), into a first sterile container. Next, group A participants (n = 33) were invited to gargle for 30 seconds with solution A, group B (n = 33) to mouthrinse for 30 seconds with solution B and group C (n = 11) to mouthrinse for 30 seconds with solution C and then to spit the solution. Five minutes later, saliva collections were done again in a second sterile container. Each cup held patient's name and the date of saliva collection while contaminated waste was appropriately discarded. Each collected sample was then inserted into separated tubes containing 2 mL of the virus transport medium (VTM) and transported to the COVID unit Laboratory in the Lebanese university for PCR processing. Reactions were performed in 20 µL final volume reaction containing 5 µL of extracted RNA, rRT-. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.07.21252302 doi: medRxiv preprint PCR was performed using CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and Bosphore Novel Coronavirus (2019-nCoV) PCR Detection Kit v4 (Anatolia, Turkey), which targeted the RdRP, N and E genes of SARS-CoV-2. In this assay, a RNase P gene region is used as an endogenous internal control for the analysis of biological samples. It is normally used to ensure the quality of the test, at extraction and PCR levels and to exclude the false negative results. Thus, in order to evaluate possible variability in the amount of material retrieved from saliva specimen before and after mouth wash we utilized RNase P as reference gene to normalize the input data. To compare the paired samples before and after mouth wash, we calculated a Ct value modified according to the ratio of sample RNase P and mean RNase P Ct values. 31 sample SarsCoV2 Ct value x sample RNaseP Ct mean RNaseP Ct value Statistical Analysis. The analysis of data was carried out using GraphPad Prism version 5 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). Differences between the means of the two matched groups were explored using Paired t test. The means of the two independent Groups were compared by using the Unpaired t test. A probability value of less than 0.05 was regarded as statistically significant. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 17, 2021. ; In total, while 77 patients were eligible for the study, 16 were excluded resulting in 61 compliant participants (Figure 1 ). Among the final study group, 36 The expression of the SARS-CoV-2 target genes (RdRp, E and N) used was approximately the same in each tested sample. To simplify our analysis, we presented the results with RdRp. For this gene the mean Ct value was 28.9 ± 5.5 (Median 29.9 [16. 45-38.16 ]) in salivary pre-wash samples. After normalization, the SARS-CoV-2 mean Ct value was 28.3 ± 6.3. Our results showed a significant mean difference between the paired samples before (29.88 ± 6.2; median 30.75) and after mouthwash (34.36 ± 6.3; median 34.19) with 1% Povidone-iodine (p < 0.0001). After mouthwash, the difference between means was 4.45 ( Figure 2A ). In addition, a higher significant difference of means was found in paired samples using Chlorhexidine 0.2% (p < 0.0001). The mean Ct increased 5.69 after mouthwash. The mean Ct of pre and post mouthwash was respectively 27.69 ± 7.16 (median 27.11) and 33.9 ± 7.08 (median 33.13) ( Figure 2B ). In contrast, no significant difference was found in the control group using the distilled water as mouthwash solution as shown in figure 2C (p = 0.566) . Moreover, the comparison of the delta Ct showed a significant difference between distilled water wash and each of the 2 solutions Povidoneiodine and Chlorhexidine 0.2% (p value 0.012 and 0.0024 respectively). No significant difference . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.07.21252302 doi: medRxiv preprint was found between the delta Ct of patients using 1% Povidone-iodine and Chlorhexidine 0.2% solutions (p = 0.24) (Figure 3 ). We noted that Ct values are considered inversely related to viral load and may serve as an indirect method of arbitrarily quantifying the viral load in the sample. With the novel COVID-19 pandemic, dental care practitioners were in urge to develop quick infection control policies. 32 So far, clinical implementation of new concepts was mainly based on recommendations without being evident based. Particularly, different pre-procedural mouthwash solutions to minimize the SARS-CoV-2 transmission during dental treatment were recommended by some dental specialist societies. 33-35 Despite lack of any clinical data supporting the virucidal effects of mouthwash solutions against SARS-CoV-2, many propositions were adopted in reviews discussing the COVID-19 preventive measurements in Dentistry. [36] [37] [38] [39] [40] [41] A recent in-vitro study tested the effect of the following mouth rinses on cell viability: hydrogen peroxide, povidone-iodine, chlorhexidine gluconate and essential oils with alcohol. The experiments found that mouth rinses can significantly reduce virus infectivity, suggesting a potential benefit for reducing SARS-CoV-2 spread. The study concluded that the clinical investigation of antiviral effects of mouth rinses is needed for proving their potential to reduce the virus spread. 42 For our knowledge this current in vivo study is the first large scale controlled-clinical trial testing the efficacy of 1% Povidone-iodine oral gargle and 0.2% Chlorhexidine oral mouthwash on salivary SARS-CoV-2 virus of positive tested patients. This study comprised 61 compliant COVID-19 positive subjects. During sampling recruitment, the non-detection of SARS-CoV-2 in nasopharyngeal samples of four hospitalized positive patients could be explained by the fact that the nucleic acid test results of a significant proportion . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.07.21252302 doi: medRxiv preprint of patients are "false negative". 43 For ethical issues, mainly avoiding the patient subsequent discomfort, we preferred to exclude these participants instead of repeating the nasopharyngeal swab test for a clinical trial purpose. In placebo-controlled clinical trials with "very ill" subjects it is unethical to assign equal subjects to each arm and it is preferable to have more subjects in test group compared to the control one. In such cases, sample size is adjusted if clear and clinically meaningful inputs on some points are available prior to working on sample size estimation. 44 As COVID-19 is a recently emerging pandemic without previous related data in addition to its critical incompletely explored status, we considered that our sample size for the test and control groups were legible. Saliva sampling was self-performed by the patients to reduce the risk of nosocomial SARS-CoV-2 transmission to health care providers. 12,45 A lapse of time of 5 minutes between mouthrinsing or gargling and second saliva collection was chosen conforming to the real procedure at dental clinic: during dental appointment, it takes usually few minutes between the patient's preprocedural . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 17, 2021. ; . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 17, 2021. ; The Ct values of RdRp obtained with RT-PCR that were detected in salivary specimens before and after mouthwash and normalized to the internal control RNAseP endogenous human gene. (A) 1% Povidoneiodine (n = 25), (B) Chlorhexidine 0.2% (n = 27) and (C) distilled water (n = 9) were used as mouthwash solutions. The before-after graph shows the changes of Ct after mouth wash for each patient. The box plots show the medians (middle line) and the first and third quartiles (boxes). The mean is marked by a plus sign inside the box. Paired Groups were compared by using the Paired t test. ***Indicates a p-value ≤ 0.0001, ns indicates no significant difference (p > 0.05). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Funding: This work was supported by the Lebanese University. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 17, 2021. ; A Novel Coronavirus from Patients with Pneumonia in China Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China The transmission and diagnosis of 2019 novel coronavirus infection disease (COVID-19): A Chinese perspective The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents 2019-nCoV transmission through the ocular surface must not be ignored Effect of cooling water temperature on the temperature changes in pulp chamber and at handpiece head during high-speed tooth preparation. Restor Dent Endod Quantitative analysis of bacterial aerosols in two different dental clinic environments Efficacy of preprocedural mouthrinses in the reduction of microorganisms in aerosol: A systematic review Reducing bacterial aerosol contamination with a chlorhexidine gluconate pre-rinse The action of three antiseptics/disinfectants against enveloped and nonenveloped viruses Drug discovery: a history Virucidal activity and cytotoxicity of the liposomal formulation of povidone-iodine Inactivation of human viruses by povidoneiodine in comparison with other antiseptics Inactivation of SARS coronavirus by means of povidoneiodine, physical conditions and chemical reagents Povidone-iodine hand wash and hand rub products demonstrated excellent in vitro virucidal efficacy against Ebola virus and modified vaccinia virus Ankara, the new European test virus for enveloped viruses Rapid and Effective Virucidal Activity of Povidone-Iodine Products Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Modified Vaccinia Virus Ankara (MVA) Prevention of hospital-acquired pneumonia (strategies for prevention of hospital-acquired infections) 2020) ADA adds frequently asked questions from dentists to coronavirus resources COVID-19 Guide pratique à partir des recommandations d'experts validées COVID-19): Implications for Clinical Dental Care COVID-19 Transmission in Dental Practice: Brief Review of Preventive Measures in Italy Approaches to the management of patients in oral and maxillofacial surgery during COVID-19 pandemic Coronavirus Disease 2019 (COVID-19): Emerging and Future Challenges for Dental and Oral Medicine A detailed report on the measures taken in the Department of Conservative Dentistry and Periodontology in Munich at the beginning of the COVID-19 outbreak Overview of transnational recommendations for COVID-19 transmission control in dental care settings Differential effects of antiseptic mouth rinses on SARS-CoV-2 infectivity in vitro Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19 Sample size estimation in clinical trial Oropharyngeal Secretion as Alternative for SARS-CoV-2 Detection Saliva as a Noninvasive Specimen for Detection of SARS-CoV-2 Saliva as an Alternate Specimen Source for Detection of SARS-CoV-2 in Symptomatic Patients Using Cepheid Xpert Xpress SARS-CoV-2 Is povidone iodine mouthwash effective against SARS-CoV-2? First in vivo tests Povidone-Iodine Use in Sinonasal and Oral Cavities: A Review of Safety in the COVID-19 Era Consideration of povidoneiodine as a public health intervention for COVID-19: Utilization as "Personal Protective Equipment" for frontline providers exposed in high-risk head and neck and skull base oncology care Preanesthetic Povidone-Iodine gargles for patients with COVID-19 Rapid In-Vitro Inactivation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Using Povidone-Iodine Oral Antiseptic Rinse Efficacy of Povidone-Iodine Nasal and Oral Antiseptic Preparations Against Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) Considerations for povidone-iodine antisepsis in pediatric nasal and pharyngeal surgery during the COVID-19 pandemic Clinical Significance of a High SARS-CoV-2 Viral Load in the Saliva Virucidal Efficacy of Different Oral Rinses Against Severe Acute Respiratory Syndrome Coronavirus 2 Is the oral cavity relevant in SARS-CoV-2 pandemic?