Electrolyzed water for the microbiologic control in the pandemic dental setting: a systematic review

Background Electrolyzed water has brought recent attention due to its antimicrobial properties. Indeed, electrolyzed water has been proposed to sterilize dental materials and instruments without compromising their structural integrity. In addition, electrolyzed water has been proposed as a mouthwash to control bacterial and viral oral infections without detrimental effects on the oral mucosa. However, no current consensus or evidence synthesis could indicate its potentially favorable use in the dental setting, particularly during the COVID-19 context. Therefore, this systematic review aimed to elucidate whether electrolyzed water could improve microbiologic control in the COVID-19 pandemic dental setting. Methods MEDLINE via Pubmed, EMBASE, Cochrane’s CENTRAL, Scopus, LILACS, and Web of Science databases were searched up to September 2021 to identify experimental studies utilizing electrolyzed water for eliminating microorganisms in a dental setting. Besides, a manual and a grey literature search were performed. The data selection and extraction were performed individually and in duplicate. The Risk of Bias (RoB) was assessed with the Nature Publication Quality Improvement Project (NPQIP) score sheet. The study protocol was registered at PROSPERO CRD42020206986. Results From a total of 299 articles, 63 studies met the inclusion criteria. The included studies assessed several types of electrolyzed waters, which showed a high disinfection potential when used to deal with different oral conditions. Electrolyzed water demonstrated a broad antimicrobial spectrum and was highly efficient in the dental office disinfection against viruses, fungi, and bacteria, being compatible with most dental materials. In addition, electrolyzed water could protect against SARS-CoV-2 infection and contamination in the dental office. Regarding the RoB, only 35.18% of entries were answered as ‘Yes’, thus achieving less than half of the reporting sheet. Conclusion Electrolyzed water effectively disinfects contaminated surfaces, dental materials, and equipment. Therefore, their use is recommendable in the SARS-CoV-2 pandemic dental setting. Supplementary Information The online version contains supplementary material available at 10.1186/s12903-022-02528-0.

COVID-19 in over 200 countries, with stunning contagion and mortality rates [1]. Recently, the coronavirus' genomic mutations and resulting variants have increased its virulence and infective potential, facilitating zoonotic and human transmission [2,3].
The main transmission route of the virus is via the release of saliva droplets or aerosols by COVID-19-infected people by coughing, sneezing, talking, or touching contaminated surfaces without adequately washing hands. Accordingly, significant loads of SARS-CoV-2 RNA have been detected in saliva [4], salivary glands [5], and oral epithelial cells [6]. Indeed, the SARS-CoV-2 virus can colonize the oral mucosa by the differential expression of angiotensin-converting enzyme II (ACE2), the SARS-CoV-2 main receptor and cell-invasion route [6]. Therefore, considering the importance of the oral cavity as the primary source of viral transmission of COVID-19, the conditions of clinical dental care should be a focus of attention. Certainly, the length of dental procedures and the proximity between patients and operators within the dental setting put dentists and their patients at high risk of COVID-19 [7]. Moreover, the constant use of water-cooled highspeed rotary hand-pieces or ultrasonic scalers during most dental procedures generates and further spreads virion-loaded aerosols [8,9]. Indeed, these aerosols can contaminate surfaces, dental instruments, impression casts, and dental unit-waterlines, among others, increasing the risk for cross-contamination [8,9].
Several disinfectants and cleaning solutions are currently available for microbiologic control at the dental office, including viruses. However, most of them are not entirely innocuous for humans, dental instruments, materials, and the environment and are not fully effective in controlling the virus dissemination [10,11]. From this basis, the use of electrolyzed water has brought recent attention due to its bactericidal and virucidal activities and less detrimental effects on biological tissues, skin, and mucosa [12]. Its antimicrobial properties have allowed it to sterilize instruments, dentures, and even impressions, without compromising their delicate structural integrity [13]. Furthermore, it has been hypothesized that its use as a mouthwash could be innocuous for the oral mucosa [14]. Thus, it could effectively reduce the SARS-CoV-2 viral load in patients' saliva before aerosol-generating procedures. However, nowadays, there is no current consensus or evidence synthesis that could indicate its potentially favorable use in the dental setting. Therefore, this systematic review aimed to elucidate whether electrolyzed water could improve the microbiologic control in the COVID-19 pandemic dental setting.

Study protocol
The protocol for this systematic review was constructed in agreement with the recommendations made by the PRISMA 2020 checklist [15] and following the quality standards recommended by the AMSTAR-2 critical appraisal tool [16]. All the authors revised, discussed, and approved the protocol for the data selection and extraction, risk of bias assessment, and data analysis a priori. Protocol registration can be found at PROSPERO CRD42020206986.

Eligibility criteria
To answer the research question: Does electrolyzed water improve microbiologic control in the dental setting? Publications that met the following PICO format and inclusion criteria were included.
• P: Oral microorganisms including viruses, bacteria, and fungi cultivated in vitro, inoculated in an animal model, or sampled from the oral cavity. • I: Electrolyzed water produced by electrolysis of regular or distilled water with any concentration of sodium chloride. • C: No intervention or other disinfectant solution used in the dental setting. • O: Colonies count, plaque reduction, infection potential, and viral RNA copies.
Due to the relative novelty of the revised topic in the dental setting, randomized clinical trials were scarce. Thus, all experimental studies were considered, including randomized or non-randomized controlled trials, animal studies with a control group, and in vitro studies. Case reports, editorial letters, and incomplete studies were excluded. Language, year, and publication status were not considered as exclusion criteria. The search retrieved articles published up to September 2021, and eligible publications in languages different from English, Spanish, French, or Portuguese were translated.

Information sources and search strategy
One author (EAC) performed the electronic search in MEDLINE via Pubmed, EMBASE, Scopus, Web of Science, Cochrane's CENTRAL, and LILACS databases, using individually adapted search strategies (Supplementary Material 1). In addition, OpenGrey and PQDT-ProQuest databases were searched for grey literature. Moreover, a manual search including recently published records from 2021 was performed in the journals from which initial studies were selected: International Journal of Oral Biology, International Journal of Clinical

Data selection and extraction
Two authors (AC and VC) performed the data selection and extraction independently. After duplicate removal, titles and abstracts were assessed for their potential inclusion. Then, full-text articles were analyzed against the inclusion criteria for their final inclusion. When disagreements occurred, inclusion was discussed with a third author (RV) until consensus. Excluded articles and their respective reasons for exclusion were recorded (Supplementary Material 2). Afterward, the following data were extracted from the included studies: Authors, year, and study setting (for reference), type of study, microorganism (source, concentration, and inoculation via), type of hydrolyzed water (concentration and preparation), duration of intervention, dental setting and comparator, effect over microorganism (time frame), and funding source, if available. Disagreements were solved by discussion, and all the extracted data were further revised by two authors (EAC and RV) to ensure the extraction of all the relevant data. In the case of missing data, the manuscripts' corresponding authors were asked for additional information.

Outcome measures
In order to evaluate the potential use of electrolyzed water for microbiologic control in the dental setting, the primary outcome measure was the inhibition of bacterial growth by means of remaining colony forming units (CFU) or viral replication by means of remaining viral RNA copies.

Risk of bias assessment
The risk of bias (RoB) was assessed independently and in duplicate by two reviewers (AC and EAC), previously calibrated in RoB assessment rounds, and the disagreements were resolved by consensus. The in vivo and in vitro studies were assessed using a modified version of the Nature Publication Quality Improvement Project (NPQIP) score sheet.

Data synthesis
Due to the substantial heterogeneity between studies, a qualitative synthesis of results was performed.

Data selection
The initial electronic search yielded 299 articles across the revised databases. Additionally, the complementary manual search resulted in 32 additional records. After duplicates were removed, 290 articles were screened. Then, revising titles and abstracts excluded 218 records, resulting in the assessment of 72 potential articles, including nine articles translated from Japanese, Korean, and Turkish. Finally, the full-text assessment resulted in the inclusion of 63 studies (Fig. 1). The main reasons for excluding full-text articles were: not being primary studies, using another disinfectant different from electrolyzed water, being incomplete, not being executed in the dental setting, and not evaluating oral microorganisms. All the included studies were in vitro except for five studies [17][18][19][20][21] which were randomized clinical trials.

Disinfection potential of electrolyzed water for oral diseases
Different varieties of electrolyzed water have been proven to be potentially helpful in treating oral diseases. In the case of caries, patients rinsing with 'electrolyzed hydrogen water' showed significantly diminished counts of Streptococci CFU in comparison with rinsing with tap water, leading to the authors' concluding that oral rinse with electrolyzed water could be supportive in diminishing the levels of caries-associated bacteria such as Streptococcus mutants [17]. Otherwise, in periodontal diseases, electrolyzed water has been tested in three randomized clinical trials. For instance, in periodontally healthy individuals, irrigation with 'aqua oxidizing water' after discontinuing oral hygiene was effective for plaque control and preventing gingivitis [19]. Likewise, the use of 'acid water' rinse inhibited the development and formation of bacterial plaque, similar to chlorhexidine in human dentine [21].
Apart from that, 'superoxidized water' irrigation along with scaling and root planning was effective for treating periodontitis-affected patients, showing diminished probing pocket depth and gingival bleeding 30 days after treatment [20].
Apart from that, the canal-disinfection potential of electrolyzed water has particularly been considered in endodontics. Indeed, a randomized clinical trial showed no differences between the use of 'oxidative potential water' in comparison with 1% NaOCl, in terms of their effectiveness in reducing the bacterial load in canals of necrotic pulpectomized primary teeth [18]. In particular, the antimicrobial properties of electrolyzed water against Enterococcus faecalis in canals of extracted teeth have been tested in seven studies [20,[22][23][24][25][26][27], being effective in reducing E. faecalis levels as an endodontic irrigant, in a similar manner to sodium hypochlorite solution ( Table 1).

Innocuity of electrolyzed water in vitro and for human application
The innocuity of electrolyzed water has been tested using in vitro models and randomized clinical trials [17][18][19][20][21]. Indeed, daily oral rinse, irrigation, or application of 'electrolyzed water' reported no adverse effects on patients [17][18][19][20][21], while having an outstanding plaque-controlling effect. Besides, canal irrigation of necrotic primary teeth on child patients with 'oxidative potential water' reported no side effects, thus considering it a harmless alternative for irrigation after pulpectomies [18]. Moreover, electrolyzed water was not cytotoxic to cultured human bone marrow-derived mesenchymal stem cells [28].

Oral microorganisms growth inhibition by electrolyzed waters
The antibacterial properties of electrolyzed water were tested in 32 in vitro studies [17,22, (Table 2). In                  (Table 4).

Compatibility of electrolyzed waters for dental materials
Some studies tested the disinfection potential and material compatibility of electrolyzed water with dental materials [33,68,[70][71][72]. For example, electrolyzed water effectively disinfected impressions made from polyvinyl siloxane [68] and alginate [69,70] without compromising their dimensional integrity or surface accuracy. Apart from that, the antimicrobial use of electrolyzed water was also demonstrated to be harmless to acrylic resin dentures contaminated with bacteria such as S. aureus [33,71,72].

Risk of bias assessment
The RoB and quality of reporting assessment using the NPQIP tool led to a total of 756 entries (Additional file 3). Of them, only 35.18% were answered as 'Yes' , while 48.15% as 'No' and the rest were marked as 'NA' (Not applicable). The tool sections ' Animal information' and 'Reagents (in vivo)' did not apply to the included studies. All of the studies scored 'Yes' in items #1 and #2 regarding sample size and collection specification. Items #3 and #4 regarding experiment replications and the use of uncommon statistical tests showed similar results with 52.46 and 45.90% of positive answers, respectively. Only one study was rated as 'Yes' in item #5, regarding the specification of using one or two-sided t or z tests. Most articles answered items #6, #7, and #8, about using P values and estimates as medians or averages, as 'Yes' , being 59.02, 68.85, and 55.73%, respectively. However, items #9-#12 (62.29, 75.40, 63.93, and 50%) mainly were answered as 'No' due to articles not reporting the definition of error bars, not assessing data normality, not calculating variation within groups and not analyzing the variance. In addition, most articles did not specify how the sample size was obtained, resulting in items #13 and #14 with 85.24 and 81.96% of 'No' answers. In this sense, items #15, #16, and #17, regarding exclusions, items #18 and #19, regarding randomization, and items #20 and #21, regarding blinding, were mostly (> 80%) answered as 'No' . Apart from that, most articles specified if their antibodies were profiled and the cell lines used, being items #22 and #23 answered as 'Yes' in 83.61 and 65.57%, respectively. Although, no articles specified if the cell lines were authenticated or if they were tested for mycoplasma contamination in items #24 and #25. Finally, most articles answered 'Not applicable' for the 'Human studies' items #26-30. In this sense, the few human studies included did identify a committee approving the protocol and the use of informed consent, but did not have clinical trial registration numbers nor used the CONSORT checklist.

Discussion
Electrolyzed water is obtained from the combination of tap water with a sodium chloride-derived solution passed through an electrolysis chamber, and it has been proven to be a cheap and environment-friendly alternative to              The water purification system using neutral electrolytic water was effective to control the proliferation of bacteria and could maintain a hygienic environment in DUWLs. disinfect different types of contaminated surfaces and inert materials due to its vast microbicide properties [53].
In the present revision, the included studies assessed several types of differently obtained electrolyzed waters, which showed a high disinfection potential when used to deal with different oral conditions, including caries, periodontal disease, and root canal system infection. These beneficial effects were achieved by reducing the amount of CFUs of disease-specific bacteria, which was higher than tap water or saline solution, reaching a disinfection potential similar to that described for chlorhexidine or sodium hypochlorite solution.
Different antimicrobial agents have been tested to reduce the cross-infection potential during dental procedures, but many of them are not completely innocuous for dental materials or safe for oral tissues [68]. In the herein included studies, electrolyzed water was demonstrated to have a broad antimicrobial spectrum and to be highly efficient in the dental office disinfection against viruses, fungi, and bacteria, being also compatible with most dental impression materials, gypsum, resin-derived materials, and dental implants. Moreover, different kinds of electrolyzed waters have been put into dental unit water lines connected to high-speed and low-speed hand-pieces, ultrasonic cavitrons, and air sprays, showing an effective capacity to control microorganisms and being harmless to oral tissues. In fact, six of the included studies showed that electrolyzed water does not produce adverse effects in patients when used as a daily mouth rinse, nor produce cytotoxic effects on cultured human bone marrow-derived mesenchymal stem cells [17][18][19][20][21]28].
These critical findings must be considered in the context of the COVID-19 pandemic since dentistry has become one of the health fields with the most significant impact on its transmissibility [73]. Indeed, the main route of airborne transmission and the consequent possible viral dissemination during dental practice originated mainly through the aerosolization produced by high-speed hand-pieces and ultrasonic scalers [63][64][65][66][67]. In this context, electrolyzed water has also shown to have great microbicidal potential against viruses that are easily transmitted during dental care, such as the human hepatitis B virus, human immunodeficiency virus, herpes simplex virus type 1, and poliovirus type 1, in a time-dependent manner [47,61,62]. In addition, the effectivity of electrolyzed water against the SARS-CoV-2 virus has also been tested in vitro, showing promising results without showing cytotoxicity [32]. Indeed, the SARS-CoV-2 virus exposed to undiluted electrolyzed water for 60 seconds significantly reduced its infectivity and replication [32]. Furthermore, oral and nasal rinse with electrolyzed water has also been shown to be protective against COVID-19 infection in a clinical trial [74]. In this study, only 1.2% of the study group that rinsed with electrolyzed water reported COVID-19 incidence, while 12.7% of the control group that did not rinse with electrolyzed water became infected [74]. The effectivity of electrolyzed water has also been proved against enveloped and non-enveloped viruses by breaking chemical bonds and changing surface proteins, mainly through its redox potential, compromising the viral envelope, viral enzymes, and viral nucleic acids [61]. Therefore, among other viruses, electrolyzed water could protect against SARS-CoV-2 infection and contamination in the dental office.
In this context, mouthwashes are commonly used as a daily antiseptic complement to toothbrushing for the maintenance of oral hygiene; however, there is still no clear evidence of their long-term effects on the healthcompatible oral microbiota [19,21]. In this sense, electrolyzed water has also been proven to be safe and significantly inhibit the growth of Streptococci in saliva, compared with saliva from patients that rinsed with tap water [76]. Indeed, the common active components of electrolyzed water, independent of their varying pH, chlorine, hypochlorous acid, and hypochlorous acidic ion, are thought to be responsible for electrolyzed water's antibacterial capacities [29].
On the other hand, fungicides are not commonly used for disinfecting the dental office, but when used, they tend to leave residues and are not entirely environmentally friendly. Likewise, in the analyzed studies, the antifungal activity of electrolyzed water was evaluated as an alternative. Nine studies showed that electrolyzed effectively inhibited the growth of C. albicans [29, 31, 34-36, 47, 52, 59, 60]. In this context, electrolyzed water antifungal activity relies mainly on OH, which causes structural damage to cell walls, leading to the leakage of K + and Mg + 2 ions and consequently compromising fungi cell function. Besides, its effectivity may also depend on its available chlorine concentrations and the thickness of the fungi cell wall [77]. Even so, other disinfectants, such as sodium hypochlorite, are more time-efficient than electrolyzed water [29].
One of the limitations of our study was the substantial methodological heterogeneity found among the included studies, which impeded the adequate pooling of results and the performance of a meta-analysis. The most common sources of heterogeneity were the different ways to prepare electrolyzed water, the diversity of the assessed pathogens, and the different exposure times. In addition, sample sizes and the adequacy of their calculation were not considered during data extraction and drawing conclusions. Indeed, normality of data distribution, sample size calculation, and error bars definition were frequently unreported. Moreover, most of the included studies had an in vitro design, which rarely reported the randomization of interventions or the blinded assessment of results, thus limiting precise clinical conclusions. However, our vast search strategy and identification of articles written in languages different from English make our study publication bias low; therefore, probably capturing most of the state-of-the-art literature regarding the use of electrolyzed water for microbial control in the dental setting. Besides, our assessment of the risk of bias among the studies suggests a cautious interpretation of the included study results.

Conclusion
The antimicrobial properties of electrolyzed water are diverse and their effective ability to disinfect contaminated surfaces, dental materials, and equipment make their use recommendable in the SARS-CoV-2 pandemic dental setting.