Reduction of SARS-CoV-2 viral load in saliva after rinsing with mouthwashes containing cetylpyridinium chloride: a randomized clinical study

Ethical approval

The research was conducted according to the Declaration of Helsinki, revised in 2013. An informed consent form describing the study objectives and risks and benefits was signed by each patient.

Trial design

This was a randomized, double-blind, single-center clinical trial. Two parallel intervention groups and one control group were designed to detect the equivalence of interventions in reducing the SARS-CoV-2 viral load in saliva.

Patient eligibility

Between April 2021 and May 2021, COVID-19 inpatients at the Hospital Israelita Albert Einstein (HIAE) were considered for participation in this investigation. The study was registered at clinicaltrials.gov (NCT04902976) in May 2021. Recruitment was initiated in April 2021 after HIAE Ethical Committee approval (43550721.4.0000.0071). The study was initiated before it was registered at clinicaltrials.gov due to the difficulties in recruiting the total number of patients needed during the COVID-19 pandemic and the oscillations of hospitalizations during this period.

The inclusion criteria were age 18 to 90 years old; up to 3 days of hospitalization length and a maximum of 7 days from first symptom appearance; positivity for SARS-CoV-2 as determined by reverse transcription-polymerase chain reaction (RT–PCR) performed in nasopharyngeal samples; positivity for SARS-CoV-2 in the first collection of saliva samples; and needing oral hygiene guidance and other preventive and therapeutic oral care. The exclusion criteria were absence of SARS-CoV-2 positivity detected by RT–PCR at the time of recruitment or at the time of the first saliva collection; oral lesions that contraindicated the use of cetylpyridinium chloride mouthwash; pregnant and breastfeeding women; bleeding in the oral cavity; allergy, irritations or other side effects derived from use of the intervention solutions; exposure to intervention solutions or other antimicrobial agents 48 h before the baseline collection; disagreement with the protocol or inadequate intervention performance by the patient.

Interventions

The following interventions were used in the study:

(a) CPC+Zn: 0.075% cetylpyridinium chloride, 0.28% zinc lactate, 225 ppm fluoride (Colgate-Palmolive Company, São Paulo, Brazil);

(b) CPC: 0.075% cetylpyridinium chloride, 225 ppm fluoride (Colgate-Palmolive Company, São Paulo, Brazil);

(c) Negative control: distilled water.

The volumes (20 mL) and rinsing times (once for 30 s) for the two test mouthwashes and for the negative control were as specified on the product label. Mouth washing was performed in the morning as the patient’s first oral hygiene procedure of the day. The participants were instructed not to perform toothbrushing or other oral hygiene procedures and were asked not to drink, eat, or chew gum after mouth washing and until the end of the experiment (for at least 60 min postrinsing). The patients were supervised by calibrated dentists to ensure compliance and to minimize experimental variations.

Outcomes

This study measured the effects of two antimicrobial mouthwashes on their ability to reduce the viral levels of SARS-CoV-2.

Sample size estimation

Patient sample size was determined based on previous data8. The size estimate was calculated on the standard deviation of the difference in viral load between the baseline and each experimental timepoint. A power of 90%, a two-sided significance level of 0.05, and a minimum of at least a 1-log detectable difference between each timepoint were adopted, which resulted in 30 patients in each group. Applying a 20% dropout rate, 35 patients were selected for each group.

Randomization and blinding

Computer-generated randomization was adopted for patient allocation. After distribution into the groups, a code was assigned to each patient, allowing allocation concealment that was maintained for the patients and the laboratory researchers. The code was only revealed at the conclusion of the study after the statistical analysis was completed. Allocation in each group was performed by two senior researchers. During the study project planning phase, patient recruitment and sample collection were set to start in February 2021 and end in April 2021. However, due to a delay in the implementation of the methodological steps in the patients’ assistant routine, the recruitment and sample collections began in April 2021 and ended in May 2021.

Saliva collection

Oral viral load was monitored based on saliva samples. For this purpose, patients were instructed by dentists, who were previously calibrated relative to saliva collection and transport. Unstimulated saliva was collected in accordance with our previous study (Eduardo et al., 2021). The time points for collection were before rinsing (T0), immediately postrinsing (within 5 min; T1), 30 min postrinsing (T2), and 60 min postrinsing (T3).

RNA extraction and quantitative reverse transcription PCR analysis

Quantitative reverse transcription polymerase chain reaction (qRT −PCR) was used for SARS-CoV-2 viral load quantification. The nucleic acids in the saliva samples were extracted (QIASymphony DSP Virus/Pathogen kit; Qiagen, Hilden, Germany), and the RNA amount and integrity in all the samples were checked (Nanodrop™; Thermo Fisher Scientific, Waltham, MA, USA). cDNA was generated based on the manufacturer’s instructions (XGEN Master COVID-19) and as described in our previous studies (Eduardo et al., 2021; Bezinelli et al., 2023). Extracted RNA (5 µL) was added to a mixture of enzymes, probes, oligonucleotides, buffer, and deoxynucleotide triphosphates (MIX CV19, XGEN Master COVID-19). Sequences in the genes for the viral nucleocapsid (N) and polyprotein ORF1ab and human RNase P (internal control) were amplified. Negative controls for qRT −PCR were free water-only samples used for RNA elution. Positive controls were used during RNA extraction (MEGAscript T7 Transcription kit; Thermo Fisher Scientific) and during amplification (provided by a commercial kit). Viral RNA extracted from SARS-CoV-2 culture supernatant was used to generate a standard curve by diluting the samples to concentrations of 10¹ to 10⁶ copies/mL. Thermal cycling was performed with a QuantStudio™ 6 Real-Time PCR System (Thermo Fisher Scientific) set at 45 °C for 15 min for reverse transcription, 95 °C for 2 min, 45 cycles of 95 °C for 10 s, and 60 °C for 50 s. A cycle threshold (Ct) >40 indicated that SARS-CoV-2 was undetectable. Viral load is shown in copies/mL.

Clinical data

Data were collected as previously described (Eduardo et al., 2021). Briefly, the following patient information was collected from their medical records: sex, age, type and frequency of COVID-19-related signs and symptoms, frequency of comorbidities, extent of lung lesions, and oxygen saturation. Indices of oral health (modified gingival index and plaque index) were also registered at baseline. Additionally, the oral cavity was visually examined using a flashlight to confirm the absence of lesions in the oral mucosa.

Statistical analyses

The double delta Ct formula ( 2^ΔΔCt) was used for the viral fold-reduction calculation. The values for the CPC+Zn mouthwash and the CPC mouthwash at T1, T2, and T3 were normalized with the mean values of the negative control ( ΔCt Control) and with the corresponding values at T0 ( ΔCtE). A cutoff value ≥ 2 at each time point was considered a significant reduction; (Dalman et al, 2012; Zhao, Erwin & Xue, 2018). The viral load was also calculated using the standard curve and was transformed in log₁₀.

Categorical and numerical clinical data were analyzed using the chi-square test and the Kruskal-Wallis test, respectively. Analysis of variance (ANOVA) was used for comparisons between the treatment groups. Within-treatment, the baseline (T0) was compared to T1, T2, and T3 using paired t tests. All statistical tests of hypotheses were two-sided, adopting a level of significance of = 0.05. The calculations were performed with Minitab v.18 statistical software (Minitab, LLC, State College, PA, USA).

Participant flow and recruitment

In this study, one hundred thirty-four (134) patients were screened. One hundred five (105) patients were enrolled and randomized in this study. Patients with undetectable virus levels in saliva at T0 were excluded from the final analysis study. Ninety-five (95) patients were included in the final analysis: 32 from the CPC+Zn group, 31 from the CPC group, and 32 from the negative control group (Fig. 1).

Baseline clinical data

Recorded clinical data are shown in Table 1. Most patients were men and had a median age of 49 years (range of 23–85 years). There were no statistically significant differences among the three treatment groups with respect to age or sex. The frequencies of COVID-19-related signs and symptoms were similar across the three treatments. More than 50% of the patients in each group reported taste changes, fatigue, fever, headache, cough, nasal congestion, and dyspnea. Approximately 50% of the patients had a risk factor for COVID-19 complications, with 15–19 patients per group reporting a risk factor. The most common risk comorbidity was hypertension, followed by diabetes and cardiovascular disease. The median values of oxygen saturation varied from 93% to 94%; seven patients had oxygen saturation <90%; and five required noninvasive mechanical ventilation. A high frequency of 50% extension lung lesions was also noted, but none of the patients progressed to severe COVID-19 at the time of the study. The participants showed good oral hygiene, with only two patients with visible dental plaque; one patient showed discrete gingival inflammation. These parameters confirmed the absence of oral infection and other oral lesions in all the patients who could exert some bias in the saliva collection and analysis. There were no statistically significant differences among the groups in any of the analyzed variables.

Fold reduction

Fold reductions (Fig. 2) were calculated using the mean values shown in Table 2. Rinsing with the CPC+Zn mouthwash reduced the viral load in saliva by 6.34-fold at T1, 3.6-fold at T2, and 1.9-fold at T3. Rinsing with the CPC mouthwash reduced the viral load in saliva by 2.5-fold at T1, 1.9-fold at T2, and 2.0-fold at T3. At T1, the CPC+Zn and CPC groups showed fold-reductions ≥2 in saliva. The CPC+Zn intervention also met these criteria at T2 (3.64-fold change) but just missed the criteria at T3 (1.90-fold change). Conversely, the CPC intervention just missed the criteria at T2 (1.89-fold change) but did meet the criteria at T3 (2.01-fold change).

Viral load

The mean SARS-CoV-2 viral loads (in log10) are shown in Tables 3 and 4. In the CPC+Zn and CPC groups, the viral loads were reduced at T1, T2, and T3 compared to baseline (Table 3). In the negative control, there was a trend toward an increase in the mean viral load from baseline to T3. The viral load reductions were statistically significant at T1 in the two test groups (Table 4).

Adverse events

Adverse events were not observed after the interventions.