key: cord-0901536-q7zk9scu
authors: Eduardo, Fernanda de Paula; Corrêa, Luciana; Mansur, Fernanda; Benitez, Carlos; Hamerschlak, Nelson; Pinho, João Renato Rebello; Heller, Debora; Bezinelli, Letícia Mello
title: Effectiveness of toothpastes on SARS-CoV-2 viral load in saliva.
date: 2022-03-23
journal: Int Dent J
DOI: 10.1016/j.identj.2022.03.006
sha: 7917646bff6a4b772ba4d2a9a648c662bc12e7d8
doc_id: 901536
cord_uid: q7zk9scu

INTRODUCTION: : The effect of toothpastes on viruses, such as SARS-CoV-2, is unknown. This study investigated the short-term effect of toothpastes containing antimicrobial properties in patients with COVID-19 to determine if they could reduce the SARS-CoV-2 salivary viral load. METHODS: : Hospitalized patients with COVID-19 (n = 83) were instructed to perform tooth brushing with one of three arms: a toothpaste containing 0.96% zinc (zinc oxide, zinc citrate) in a silica base (Test 1); toothpaste containing 0.454% SnF(2) in a silica base (Test 2), and a nonantibacterial toothpaste (control). Saliva was collected before intervention (T0), immediately after intervention (T1), and 30 (T2) and 60 min (T3) after intervention. The SARS-CoV-2 salivary viral load was measured using quantitative real-time polymerase chain reaction (qRT-PCR) assays. For Test 1 and Test 2 toothpastes, the fold reductions were normalized to baseline and to the control toothpaste at each time point after brushing. A fold change of ≥2 is considered clinically effective. RESULTS: : Brushing with the Test 1 toothpaste reduced the SARS-CoV-2 salivary viral load by 4.06-fold at T1, by 2.36-fold at T2, and by 1.42-fold at T3. Similarly, brushing with a Test 2 toothpaste reduced the SARS-CoV-2 salivary viral load by 2.33-fold at T1, by 2.38-fold at T2, and by 0.77-fold at T3. CONCLUSION: : Immediately after brushing, the use of antimicrobial toothpastes reduced the salivary viral load of patients with COVID-19. The trial was registered on https://clinicaltrials.gov/ (NCT04537962).

The novel coronavirus disease 2019 (COVID- 19) pandemic has mobilized the scientific community to establish antimicrobial protocols that are able to reduce or eradicate the novel coronavirus in human tissues and object surfaces. In patients with COVID-19, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is present in the oral mucosa and saliva. 1 The effectiveness of oral antiseptic products and their active ingredients against SARS-CoV-2 have been investigated in vitro [2] [3] [4] [5] [6] [7] [8] [9] and in vivo. [10] [11] [12] [13] However, only two of the in vitro studies and none of the clinical studies evaluated toothpaste formulations. A toothpaste slurry containing 0.05% cetylpyridinium chloride (CPC) produced a 3.3-log 10 reduction in viral titer as reported by Komine et al., 5 and a fluoride toothpaste slurry produced a 2.26-log 10 reduction in viral titer as reported by Shewale et al. 8 The present study examined whether toothpastes with antimicrobial properties have a potential anti-SARS-CoV-2 effect in patients with COVID-19. This research was motivated by recent investigations that revealed the SARS-CoV-2 presence in the gingival crevicular fluid, 14 leading to the hypothesis that SARS-CoV-2 viral particles participate in oral biofilm colonization in dental and periodontal tissues and that the dental plaque and saliva could be reservoirs of the novel coronavirus. 15 Stannous fluoride inhibits the bacterial glycolysis and, in conjunction with fluoride, prevents the accumulation of supragingival dental plaque and calculus and reduces gingival inflammation. 16 A stannous fluoride toothpaste stabilized with zinc phosphate has been shown to significantly improve the control of dental plaque, calculus formation, dentinal hypersensitivity, and gingivitis. 17 In a toothpaste, the zinc oxide and zinc citrate combination has been shown to produce enhanced antimicrobial effectiveness. 18 Zinc has been considered an important element for controlling COVID-19 infection due to its antiviral properties as well as immune system stimulation and oxidative stress inhibition and have primarily been observed through the use of zinc supplements. 19 Based on the antibacterial properties and potential antiviral effects of toothpastes containing metal ions, this study aimed to investigate the short-term effects of toothpastes composed by these elements on the SARS-CoV-2 salivary viral load of patients with COVID-19.

This study was approved by the Ethical Committee of Human Research of Hospital Israelita Albert Einstein, Brazil, (project no. 32018820.5.0000.0071) and was conducted according to the Declaration of Helsinki, as revised in 2013. After being informed regarding the proposed study's nature along with known benefits and risks, each patient provided informed consent.

This was a randomized, double-blind, single-center clinical trial that involved hospitalized patients with COVID-19. The study involved three parallel intervention groups with a conceptual framework to assess the equivalence of interventions to reduce the SARS-CoV-2 salivary viral load.

Patients diagnosed with COVID-19 and hospitalized in negative pressure rooms from December 2020 to May 2021 participated in the study. The inclusion criteria were both sexes, age > 18 years, SARS-CoV-2 positivity confirmed by nasopharyngeal samples After consent form signing, the patient performed the first tooth brushing of the day following the researcher's instructions. The patient received a toothbrush with soft bristles and a tongue and cheek cleaner that had been covered with a standardized quantity (1 g) of toothpaste. The toothbrush was previously prepared by the researcher and offered to the patient without toothpaste identification. The patient brushed for 2 min, applying their usual brushing technique. During this brushing, the tongue and cheek cleaner cleans the cheeks. The researcher controlled the tooth brushing time with a chronometer. After the procedure, the patient expectorated the toothpaste/saliva slurry and gently brushed the tongue dorsum using the tongue and cheek cleaner. The patient was instructed to place the tongue and cheek cleaner at the back of the tongue as far as he could comfortably reach and pull the cleaner forward to the tip of the tongue. To clean the entire tongue, this motion was repeated several times The patient was instructed to rinse the oral cavity with water and not use dental floss and mouthwash solutions during the study's evaluation periods.

The primary outcome was the fold reduction in SARS-CoV-2 salivary viral load after the interventions. This outcome was assessed by analyzing the salivary viral load reduction percentage measured by salivary quantitative reverse transcription PCR tests.

The sample size was estimated based on a previous pilot study's standard deviation. 11 For the sample size estimation, the following parameters were set: minimal detection difference of one viral log reduction, 80% power, 95% confidence interval, and 5% alpha. Based on this, 25 patients were required in each arm.

For patient allocation, computer-generated randomization was adopted. The computer also generated codes for each intervention and patient, which were accessed by the researcher who instructed the patient during the intervention and the laboratory staff.

Throughout the study, allocation concealment was maintained. Two senior researchers (FPE and LMB) were responsible for the allocation sequence, patient enrollment, and intervention assignment. Recruitment and allocation were performed to achieve each arm's estimated sample size.

The patient and the laboratory staff who analyzed the samples were blinded to the intervention until the end of statistical processing.

Before the intervention, a general clinical evaluation was performed to determine the teeth and gingiva's oral health quality. Gingival inflammation and dental plaque accumulation were recorded following the modified gingival index 20 and plaque index. 21 These indexes were chosen for being noninvasive and not requiring a periodontal probe or plaque staining, techniques that are difficult to perform at the bedside and can increase the SARS-CoV-2 contamination risk. Based on individual scores attributed to each analyzed tooth, an average score was obtained (one anterior tooth and two posterior teeth in each dental arcade).

Unstimulated saliva (1 mL) was collected before intervention (T0), immediately after intervention (T1), and 30 min (T2) and 60 min (T3) after intervention. The researcher controlled the experimental times with a chronometer. The patient was instructed to expectorate saliva into a 50-mL sterile tube for 5 min. 22 Only samples with at least 1 mL volume were included in the study. During sample collection, the samples were stored in ice and immediately sent to the laboratory for RNA extraction.

Total RNA extraction and quantitative reverse transcription PCR analysis SARS-CoV-2 viral load quantification was performed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). 23, 24 The saliva samples' nucleic acid was extracted using the QIASymphony DSP Virus/Pathogen kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. In all samples, the RNA amount and integrity was checked by Nanodrop at the moment of RT-PCR assay. To quantify the total number of viral particles present in the patient samples, a standard curve was used. The standard curve was generated by serially diluting the positive control RNA, previously described above, into a range of 10 1 to 10 6 copies/mL virus particles. Primer efficiency assays were performed using known viral cDNA standards to ensure proper amplification of the target genes. For the reverse transcription, the PCR cycling conditions were 45°C for 15 min, followed by 95°C for 2 min, then 45 cycles of 95°C for 10 s, and 60°C for 50 s. The cycles were performed using the QuantStudioTM 6 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Cases with >40 cycle threshold (Ct) value were considered SARS-CoV-2 undetectable.

Data about sex, age, COVID-19 signs and symptoms, presence of comorbidities, the extension of lung lesions, and oxygen saturation were collected from medical records.

The researcher also noted the patients' reports about dry mouth and taste/smell changes.

Fold reduction was calculated against the control toothpaste with the difference to relative baselines used to quantify the reduction of SARS-CoV-2 level in the saliva. The control was considered as reference control (CtC), and baseline time (T0) was considered the experimental control (CtE). ΔCtC was calculated by normalizing the Ct values from the control at T1, T2, and T3 with those at T0. ΔCtE was calculated by normalizing the Ct values for the Test 1 and Test 2 toothpastes at T1, T2, and T3 to the corresponding values at T0. ΔΔCt (fold-difference) was then determined using the formula 2 -(ΔCTC-ΔCTE) . The fold reduction was calculated using 1/ΔΔCt. A fold-change cutoff value ≥ 2 was specified. 25, 26 If a fold-change reduction is found to be equal to or greater than the specified cutoff, then the fold-change reduction at the time point is considered a significant reduction.

A total of 1847 patients that showed mild-to-moderate COVID-19 signs and symptoms were screened to assess study eligibility. From these patients, 83 were randomized into one of the three study arms. One, two, and five patients were excluded from Test 1, Test 2, and control arms, respectively, due to the absence of viral detection in the saliva at baseline. In total, 25 patients were analyzed in each arm ( Figure 1) . Table 1 shows the clinical data, including gingival health conditions, detected at baseline. Most of the three arms' patients were male, with a median age of 51 or 52 years and a range of 21-75 years across the study. Among the three groups, there were no statistically significant differences with respect to age or gender. Ten (10) patients in each group had comorbidities and were at risk for COVID-19 complications.

In each group, most patients exhibited fatigue, fever, headache, coughing, and dyspnea. Smell and taste changes were detected in less than half of the patients. Dry mouth was reported by at least 60% of the patients in each group, but a sufficient amount of saliva was collected from all patients. Patients using the Test 1 toothpaste had a higher abdominal pain incidence than in the other groups. For oxygen saturation, the median values were either 94% or 95% with a range of 88%-100% across all patients. There were no patients with extension of lung lesions by >50%; however, less than 50% of each group had lung lesions with extensions of up to 25%. There were no patients with caries activity and residual tooth roots. All patients had satisfactory oral health, with more than 80% of patients exhibiting no signs of gingival inflammation, and more than 70% exhibiting no visible plaque. All other patients only had mild gingivitis, and only two patients exhibited a moderate amount of plaque.

Fold reduction (Figure 2 ) was calculated using the Ct mean values shown in Table 2 .

Based on double ΔCt value, which is an approximation method to determine relative gene expression with qRT-PCR experiments, and normalized for T0 (baseline) and the control toothpaste, brushing with the Test 1 toothpaste reduced the salivary viral load by 4.06-fold at T1, 2.36-fold at T2, and 1.42-fold at T3. Similarly, brushing with the Test 2 toothpaste reduced the salivary viral load by 2.33-fold at T1, 2.38-fold at T2, and 0.77fold at T3. Both Test 1 and Test 2 toothpastes met the minimum acceptance clinical criteria of at least a 2-fold reduction in viral bioload in the saliva at T1 and T2 when normalized to the NaMFP toothpaste and the baseline. At T3, neither toothpaste met the criteria.

The patient mean values for SARS-CoV-2 viral load (log 10 ) are included as Supporting Information for all three test products at each timepoint.

None of the patients experienced discomfort or side-effects as an intervention result.

Toothpastes and their ingredients have demonstrated in vitro effectiveness against the SARS-CoV-2 virus, 5,8 the human influenza viruses, 27 and the human parainfluenza virus. 28 Using an in vitro assay, Tateyama-Makino et al. 29 determined that five surfactants, commonly found in commercially available toothpastes and mouthwashes, exhibited inhibitory effects on the SARS-CoV-2 spike protein with ACE2 interaction and on the TMPRSS2protease activity. They further postulated that these ingredients could help in the SARS-CoV-2 infection prevention.

This is the first clinical study that analyzed the potential antiviral effects of toothpastes on SARS-CoV-2 in patients with COVID-19. Both Test 1 and Test 2 toothpastes demonstrated clinically relevant fold changes relative to baseline and the NaMFP toothpaste control at both T1 and T2. Zinc is considered an important element in the control of COVID-19 infection owing to its antiviral, immune system-stimulatory, and oxidative stress-inhibitory properties. 19 In the oral cavity, besides the prevention of dental plaque accumulation and gingivitis, toothpastes containing zinc have an antiinflammatory effect, reducing the number of polymorphonuclear cells in the saliva. 30 Moreover, toothpastes composed of zinc and arginine can improve the oral keratinocyte function against pathogenic microorganisms. 31 For maintaining human microbiome homeostasis in patients with COVID-19, a good oral hygiene routine is essential, by preventing secondary intestinal and pulmonary bacterial and other viral infections. 32, 33 The oral biofilm has been implicated in COVID-19 pneumonia by aspiration of oral microorganisms and induction of dysbiotic communities in the lungs. 15 Oral microbiome changes can have a detrimental impact on oral health; thus, maintaining a balanced oral microbiome is crucial to maintaining oral health. 34 In conclusion, this study has demonstrated that brushing with antimicrobial toothpastes reduced the salivary viral load immediately after brushing in patients with COVID-19 as measured by fold changes relative to baseline and to the nonantibacterial fluoride toothpaste. This suggests that there may be merit in incorporating the toothpaste use with antimicrobial properties as part of a risk-mitigation strategy for patients infected with SARS-CoV-2. Although these toothpastes will not treat or prevent the disease, their use will temporarily reduce the viral load in the oral cavity.

Scientific rationale for study: The effect of such toothpastes on viruses, like SARS-CoV-2, is unknown.

Principal findings: Although these toothpastes will not treat or prevent the disease, their use will temporarily reduce the viral load in the oral cavity.

Practical implications: There may be merit in adding the toothpaste use with antimicrobial properties as part of a risk-mitigation strategy for patients infected with SARS-CoV-2.

The authors declare that there are no conflicts of interest.

We thank the study patients for their time and commitment to the study. We thank all the members of the clinical diagnosis laboratory at the Hospital Israelita Albert Einstein for their dedication and work that made this study possible. We thank Colgate-Palmolive 

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Modified gingival index grades: 0, absence of inflammation; 1, mild inflammation in some regions of the gingiva (marginal gingiva or papilla); 2, mild inflammation in all regions of gingiva; 3, moderate inflammation (erythema, edema, and hypertrophy); 4, severe inflammation with spontaneous bleeding, congestion, or ulceration