Effects of Mouthwash on Oral Cytomegalovirus Epstein‐Barr Virus Herpes Simplex Virus Type-1

   

Hessam Nowzari^1* and Petra Wilder-Smith²

¹Private Practice, Beverly Hills, California, USA

²Professor, Director of Dentistry; Beckman Laser Institute, University of California, Irvine; 1002 Health Sciences Rd East, Irvine, CA 92617, USA

Citation: Nowzari H, Wilder-Smith P. (2022) Effects of Mouthwash on Oral Cytomegalovirus, Epstein‐Barr virus, Herpes Simplex virus type-1. Adv Clin Med Res. 3(4):1-9.

Received: October 17,  2022 | Published: November 8, 2022

Copyright^© 2022 genesis pub by Nowzari H, et al. CC BY-NC-ND 4.0 DEED. This is an open-access article distributedunder the terms of the Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License.,This allows others distribute, remix, tweak, and build upon the work, even commercially, as long as they credit the authors for the original creation.

DOI: https://doi.org/10.52793/ACMR.2022.3(4)-44

Abstract

Background: Cytomegalovirus (HCMV), Epstein‐Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) are pathogens.

Objectives: The goal of the present double-blinded, randomized study was to compare the effect on oral viral load of twice daily use over 60 days of Lumineux Mouthwash^Rvs. de-ionized water. The main composition of the mouthwash was Dead Sea salt.

Methods: 30 participants were randomized to test or control. For 60 days, participants rinsed for 60s twice daily with 20ml of their allocated mouthwash, after morning and evening meals. On Day 0 and 60, before eating and oral hygiene and at least 60 minutes after drinking, unstimulated saliva was collected. Samples underwent mRNA analysis. Study endpoints were changes in Log Salivary Viral Load.

Result: After adjusting for baseline differences, the reduction in viral load was significantly greater for the test group, all p-values <0.001. Baseline differences did not have an effect on the differences between groups in change over time.

Conclusion: After adjusting for baseline differences, the reduction in viral load was significantly greater for the test group, all p-values <0.001. Baseline differences did not have an effect on the differences between groups in change over time.

Keywords

Cytomegalovirus; HCMV; Periodontitis; CMV; Epstein‐Barr virus

Introduction

Human cytomegalovirus (HCMV), Epstein‐Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) are emerging as major pathogens, in particular in immune compromised individuals [1]. Herpesviruses and oral bacteria may interact synergistically in causing significant infections which can be associated with severe clinical consequences.

Studies by Contreras et al. have presented strong evidence for the role of herpesviruses in the pathogenesis of human periodontal disease [3]. Contreras et al. determined the presence of herpes viruses in polymorphonuclear neutrophils, monocytes, macrophages and T and B lymphocytes in biopsies of periodontitis lesions from 20 adults. Periodontitis-derived monocytes and macrophages revealed HCMV in cell fractions from 11 (55%) patients and HSV in cells from 1 (5%) patient. T lymphocytes harbored HCMV in cell fractions from 4 (20%) patients and HSV in cell fractions from 4 (20%) patients. B lymphocytes showed EBV-1 in cell fractions from 9 (45%) patients. The study suggested that HCMV infects periodontal monocytes, macrophages and less frequently T lymphocytes and that EBV-1 infects periodontal B lymphocytes.

Our group studied active periodontitis lesions in 19 trisomy-21 patients detecting HCMV in 26% of patients [4]. In healthy periodontal sites, only one revealed HCMV. Subgingival debridement using a combination of hand and ultrasonic instruments did not reduce the presence of genomic herpesvirus. Viral-bacterial co-infections were observed in trisomy-21–associated destructive periodontal disease. We suggested that viral infection may reduce periodontal defense mechanisms and promote growth of putative periodontopathic bacteria such as Tenarellaforsythensis, Prevotella intermedia, and Capnocytophaga species. HCMV–Actinobacillusactinomycetemcomitans co-infection in localized aggressive periodontitis was reported by Nowzari et al. in a patient suffering from Fanconi anemia [5]. Nowzari et al. also analyzed HCMV pp67-mRNA amplification in oral fluids of 38 renal transplant recipients at 6 months post-transplantation [6]. Although patients had received antiviral therapy until 3 months post-transplantation, HCMV gene transcripts were detected in the saliva of 21% and the gingival crevicular fluid of 18% of patients. All patients (100%) with HCMV pp67-mRNA detected in saliva demonstrated clinical manifestations of viral infection, as did 86% of patients with HCMV pp67-mRNA detected in the gingival crevicular fluid. Transplant complications requiring urgent hospitalization were observed in 15 patients. Six of these patients were diagnosed with gingival overgrowth and active HCMV-associated periodontitis. 

HCMV, EBV, and HSV-1each occurs in about 5% of healthy periodontal sites and 50% of severe periodontitis lesions [1-2]. In progressive periodontitis sites, herpesvirus copycounts can exceed bacterial cell counts [1-2]. In 2022, Nowzari et al. evaluated the composition of Dead Sea salt, its in vitro cytotoxicity, and reported on its efficacy against oral bacterial leukotoxins, oral endotoxins and oral glucan sucrose [7]. The most predominant elements detected in Dead Sea salt were the water of crystallization (H2O, water that is found in the crystalline framework of salt and which is not directly bonded), magnesium 0 chloride (MgCl2), potassium chloride (KCl), sodium chloride (NaCl), calcium chloride (CaCl2), bromide (Br -) and sulfates (SO4). While no cytotoxicity was detected, Dead Sea salt was highly effective against leukotoxin, endotoxin, and glucan sucrase enzyme. The authors suggested that rinsing with Dead Sea salt has the potential to contribute to the prevention of oral infections and recommended clinical research.

The goal of the present single center, double-blinded, randomized in vivo study was to compare the effect on oral viral load in 30 individuals of twice daily use over a period of 60 days of Lumineux Oral Essentials Clean and Fresh Mouthwash (Oral Essentials, Beverly Hills, CA 90210) vs. de-ionized water. The main composition of the tested mouthwash is Dead Sea salt. 

Material and Method

Participants

30 individuals who met inclusion/exclusion criteria were recruited by mass e-mails and word of mouth on and around the University of California, Irvine campus. They provided written, informed consent under University of California, Irvine IRB-approved protocol # 2020-5719. Participants met the following inclusion and exclusion criteria: 

Inclusion criteria

• Male or female aged 25-35
• Gingival Index>2 [8]
• Able to provide written informed consent
• Able to attend study visits
• Available for follow up on the telephone
• Minimum of 20 teeth
• Measurable salivary viral load for HSV-1, HCMV and EBV at baseline.

Exclusion criteria

• Use of antibacterial mouth rinse within 3 months or during study
• Systemic or topical oral antibiotic, antiviral, antifungal medications within 3 months or during study
• Any dental treatment within 1 month or during study
• History of significant adverse effects following use of oral hygiene products such as toothpastes and mouth rinses or allergy to personal care/consumer products or their ingredients.
• Presence of any condition, abnormality, or situation at baseline that in the opinion of the Principal Investigator may preclude the volunteer’s ability to comply with study requirements, including completion of the study or the quality of the data.

The study was performed in full compliance with University of California, Irvine IRB protocol 2020-5719, and all clinical procedures were conducted in accordance with the Helsinki Declaration of 1975, as updated in 2013. No changes were made in the study design after commencement of the study.  Participants were randomized in a 1:1 ratio (randomizer.com) to use either the test rinse (Lumineux Oral Essentials Clean and Fresh Mouth wash, Oral Essentials, Beverly Hills, CA 90210), or a negative control rinse (de-ionized water (University of California, Irvine storehouse). Mouthwash bottles were masked to conceal the rinse’s identity from study participants and investigators. Subjects were asked to store and return all used mouth rinse containers to enable verification of usage compliance. Participants were contacted by telephone weekly to monitor and reinforce compliance. They were asked to keep up any pre-existing hand washing and mask-wearing routine, not to change other hygienic habits, and not to take any cold remedies during the intervention period. Participants maintained a daily health log, recording presence, duration, as well as any signs or symptoms that deviated from full health. This log included any un-wellness, including any upper respiratory tract infections (URTI) complaints such as nasal symptoms (rhinorrhea and sneezing), pharyngeal symptoms (soreness and scratchiness), bronchial symptoms (cough and phlegm), and general symptoms (feverishness, arthralgia, malaise, and any other deviations from full health). Each symptom was classified into four grades, that is, none (0), mild (1), moderate (2), and severe (3), according to the Jackson method. “Mild” was defined as when a participant was unaware of the symptom when he/she was busy; “moderate” as when one always felt discomfort; and “severe” as when one experienced difficulty in activities of daily life.

For 60 days, after shaking the bottle thoroughly, participants rinsed for 60s twice daily with 20ml of their allocated mouthwash, directly after morning and evening meals. They abstained from food and drink for at least 30 mins after rinsing. On Day 0, before eating and oral hygiene, before mouthwash use had begun, and at least 60 minutes after drinking, unstimulated saliva was collected. Participants were asked to accumulate saliva in the floor of the mouth and spit it out into a graduated Zymo Collection Tube^R every 60 seconds for 5 minutes, then to shake it vigorously to ensure proper stabilization. Saliva was again collected in the same way on Day 60 of the study.  Samples were frozen in an -800⁰C freezer, where they were stored until all the samples were acquired and were processed together.Saliva samples underwent mRNA analysis using RT-PCR of viral load of HSV1, CMV, and EBV (Thermo-Fischer Scientific, Waltham, Mass 02451, USA). Study endpoints included (a) changes in Log Salivary Viral Load (HS-1, HCMV and EBV) Day 60 vs Day 0, and (b) presence and severity of any illness and of URTI-specific symptoms on the health log.

Results

Participants

All participants completed the study in full compliance with the protocol. Their demographics are shown in (Table 1).  No adverse events were reported or observed.

	Gender (M/F/N)	Race	Ethnicity	Age Range	Mean Age	Median Age
Control Group	9M; 7F; 0N	8A; 6W; 2MR	4H	25-34 y	28.0 y	28 y
Test Group	8M; 6F; 0N	9A; 4W;1PI	3H	25-35 y	29.8 y	29.8 y
Total	17M; 13F; 0N	17A;10W;2MR; 1PI	7H	25-35 y	28.9 y	28 y