Clinical Oral Investigations (2020) 24:3707–3713

https://doi.org/10.1007/s00784-020-03549-1

SHORT COMMUNICATION

A prospective clinical pilot study on the effects of a hydrogen

peroxide mouthrinse on the intraoral viral load of SARS-CoV-2
Maximilian J. Gottsauner 1 & Ioannis Michaelides 2 & Barbara Schmidt 3 & Konstantin J. Scholz 4 & Wolfgang Buchalla 4 &
Matthias Widbiller 4 & Florian Hitzenbichler 5 & Tobias Ettl 1 & Torsten E. Reichert 1 & Christopher Bohr 2 &
Veronika Vielsmeier 2 & Fabian Cieplik 4

Received: 29 July 2020 / Accepted: 24 August 2020 / Published online: 2 September 2020
# The Author(s) 2020

Abstract
Objectives SARS-CoV-2 is mainly transmitted by inhalation of droplets and aerosols. This puts healthcare professionals from
specialties with close patient contact at high risk of nosocomial infections with SARS-CoV-2. In this context, preprocedural
mouthrinses with hydrogen peroxide have been recommended before conducting intraoral procedures. Therefore, the aim of this
study was to investigate the effects of a 1% hydrogen peroxide mouthrinse on reducing the intraoral SARS-CoV-2 load.
Methods Twelve out of 98 initially screened hospitalized SARS-CoV-2-positive patients were included in this study. Intraoral
viral load was determined by RT-PCR at baseline, whereupon patients had to gargle mouth and throat with 20 mL of 1%
hydrogen peroxide for 30 s. After 30 min, a second examination of intraoral viral load was performed by RT-PCR.
Furthermore, virus culture was performed for specimens exhibiting viral load of at least 103 RNA copies/mL at baseline.
Results Ten out of the 12 initially included SARS-CoV-2-positive patients completed the study. The hydrogen peroxide mouthrinse
led to no significant reduction of intraoral viral load. Replicating virus could only be determined from one baseline specimen.
Conclusion A 1% hydrogen peroxide mouthrinse does not reduce the intraoral viral load in SARS-CoV-2-positive subjects.
However, virus culture did not yield any indication on the effects of the mouthrinse on the infectivity of the detected RNA copies.
Clinical relevance The recommendation of a preprocedural mouthrinse with hydrogen peroxide before intraoral procedures is
questionable and thus should not be supported any longer, but strict infection prevention regimens are of paramount importance.
Trial registration German Clinical Trials Register (ref. DRKS00022484)

Keywords SARS-CoV-2 . COVID-19 . Hydrogen peroxide . Mouthrinse . Gargle . Intraoral . Viral load

Maximilian J. Gottsauner and Ioannis Michaelides contributed equally

and share first authorship. Introduction
Veronika Vielsmeier and Fabian Cieplik contributed equally and share
senior authorship. The coronavirus disease 2019 (COVID-2019), which is
caused by a novel coronavirus SARS-CoV-2 (severe acute
* Fabian Cieplik respiratory syndrome coronavirus 2), amounts to more than
fabian.cieplik@ukr.de 19.9 million confirmed cases and more than 730,000 attribut-
1 ed deaths, as per August 11, 2020, and thus represents one of
Department of Oral and Maxillofacial Surgery, University Hospital
Regensburg, Regensburg, Germany the greatest challenges for the whole healthcare sector in the
2 twenty-first century [1].
Department of Otorhinolaryngology, University Hospital
Regensburg, Regensburg, Germany The main routes of SARS-CoV-2 transmission are by
3 direct contact or by airborne transmission due to inhala-
Institute of Clinical Microbiology and Hygiene, University Hospital
Regensburg, Regensburg, Germany tion of aerosols and respiratory droplets [2]. This puts
4 healthcare professionals (HCPs) at high risk of nosoco-
Department of Conservative Dentistry and Periodontology,
University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, mial infection with SARS-CoV-2 [3], as it was already
93053 Regensburg, Germany shown by a disproportionally high infection rate for
5
Department of Infection Prevention and Infectious Diseases, HCPs in an early report from Wuhan, China [4].
University Hospital Regensburg, Regensburg, Germany Patients with SARS-CoV-2 infection exhibit a very high
3708 Clin Oral Invest (2020) 24:3707–3713

viral load in the oropharynx, the oral cavity, and the Material and methods
nose, irrespective of the presence of clinical symptoms
[2, 5]. Therefore, particularly HCPs from specialties with Study design
close contact to this region such as dentists, maxillofacial
surgeons, and otorhinolaryngologists are at tremendous The present study is a prospective clinical pilot study investi-
risk of being infected and becoming potential carriers gating the effects of a mouthrinse with 1% hydrogen peroxide
of the virus [6–13]. Accordingly, the first COVID-19- on the intraoral viral load of SARS-CoV-2-positive patients
related fatality of a medical doctor documented globally hospitalized at the isolation ward of the University Hospital
was that of an otorhinolaryngologist in Wuhan on Regensburg during an investigation period of 2 months from
January 25, 2020 [14]. Furthermore, clinical procedures April until May 2020.
in these specialties and particularly in clinical dentistry Only patients with a positive test for SARS-CoV-2 within
often involve generation of aerosols [15], which further the last 72 h were included in this study. Exclusion criteria
increases the risks of nosocomial infection with SARS- were indication for intubation or mechanical ventilation and
CoV-2 among HCPs [11]. severe stomatitis.
Therefore, special infection control regimens have been Patients were screened for eligibility by one medical doctor
introduced in dental practices all over the world, including (IM) and provided with detailed description of the study out-
patient triage, personal protective equipment (such as par- line which involved the following procedure: patients were
ticulate respirators based on n-95 or FFP2 standards), use asked to gargle their mouth and throat with 20 mL 0.9%
of rubber dam isolation, and general limitation of aerosol- NaCl for 30 s for acquiring a baseline oropharyngeal specimen
generating procedures [7–9, 11, 16, 17]. Following a first for the SARS-CoV-2 real-time PCR (RT-PCR) test.
recommendation by Peng et al. [12], some author groups Immediately afterwards, patients had to perform a mouthrinse
also proposed to let patients perform preprocedural with 20 mL 1% hydrogen peroxide by gargling their mouth
mouthrinses with oxidizing agents such as 1% hydrogen and throat for 30 s. Thirty minutes after this mouthrinse, an-
peroxide or 0.2% povidone iodine in order to reduce the other oropharyngeal specimen for the SARS-CoV-2 RT-PCR
intraoral viral load before conducting any intraoral proce- test was acquired by letting the patients gargle their mouth and
dures [7–9, 17, 18]. Accordingly, the American Dental throat with 20 mL 0.9% NaCl for 30 s. The respective quan-
Association suggested on their website on March 12, tities of copies/mL of SARS-CoV-2 RNA were analyzed by
2020, to have patients gargle with a 1% hydrogen peroxide RT-PCR.
solution before each appointment [19]. Likewise, this sug- Written informed consent was obtained from all individual
gestion was also disseminated among German dentists by participants included in the study. The study design was ap-
the Institut der Deutschen Zahnärzte in their brochure proved by the ethics committee of the University of
about recommended standard dental procedures during Regensburg (ref. 20-1787-101) in accordance with the 1964
the COVID-19 pandemic from April 24, 2020 [20]. Helsinki declaration and its later amendments or comparable
The recommendation of a preprocedural mouthrinse ethical standards. The study has been registered at the
with hydrogen peroxide was mainly based on the general German Clinical Trials Register (ref. DRKS00022484).
vulnerability of SARS-CoV-2 toward oxidation [12], and
on the finding that products containing oxidizing agents RT-PCR-based analysis of viral load
such as hydrogen peroxide and povidone iodine were able
to inactivate coronaviruses on inanimate surfaces within a Nucleic acids were extracted from 200 μL of oropharyngeal
1-min exposure period [21, 22]. However, until now, there specimens using EZ1 Virus Mini Kit v2.0 in combination
are no clinical data supporting the efficacy of suchlike with the EZ1 Advanced XL system (Qiagen, Hilden,
preprocedural mouthrinses in terms of reducing the Germany), as recommended by the manufacturer. Viral
intraoral viral load in SARS-CoV-2-infected patients RNA was amplified and detected in duplicate using the
[23–26]. Due to the high viral load in the oropharynx and SARS-CoV-2 E gene RT-PCR [29] on the StepOnePlus
the oral cavity, there may be some recontamination soon RT-PCR System (ThermoFisherScientific, Schwerte,
after performing the mouthrinse [27]. Furthermore, caution Germany). Bacteriophage MS2 was used as internal control
should be taken before generally recommending antimicro- to check for extraction and amplification efficacy [30].
bial mouthrinses due to the inherent risks of inducing det-
rimental shifts in the oral ecosystem [28]. Virus culture
Therefore, the aim of this clinical pilot study was to
investigate the effects of a mouthrinse with 1% hydrogen SARS-CoV-2 was isolated from the oropharyngeal specimens
peroxide on the intraoral viral load in SARS-CoV-2- that exhibited more than 103 copies/mL of SARS-CoV-2 RNA
positive patients. at baseline by using kidney epithelial cells from African green
Clin Oral Invest (2020) 24:3707–3713 3709

monkey (Vero-CCL19 cells, ATCC). Cells were cultivated in hospitalized in an intensive care unit (without need of intuba-
Dulbecco’s Modified Eagle‘s Medium supplemented with 10% tion), and 11 were hospitalized in an isolation ward. Eleven
heat-inactivated fetal calf serum (Sigma-Aldrich, Munich, out of the 12 patients showed comorbidities (e.g., diseases of
Germany), 90 U/mL streptomycin, 0.3 mg/mL glutamine, the liver, cardiovascular system or kidney, hematological dis-
200 U/mL penicillin, and 2.5 μg/mL amphotericin B (PAN eases, and obesity). Only one of the patients presented no
Biotech, Aidenbach, Germany). After inoculation of oropha- symptoms of the infection with SARS-CoV-2. The time peri-
ryngeal specimens for 24 h, cells were washed twice before od between diagnosis of SARS-CoV-2 infection and inclusion
viral loads in the supernatants were determined 7 days post- in the study ranged from 1 to 5 days (median 3 days). Table 1
infection by RT-PCR as described above. shows a detailed overview of the patient population included
in this study. Figure 1 shows the flow of patients through the
Data analysis stages of this study.

Data are reported as median values (with 1st and 3rd quartiles) RT-PCR-based analysis of viral load prior and after 1%
and were statistically analyzed non-parametrically using the hydrogen peroxide mouthrinse
Wilcoxon signed-rank test for related samples on a signifi-
cance level of α = 0.05. All statistical analyses were per- In two out of the 12 initially included patients, no SARS-CoV-
formed using IBM SPSS Statistics, version 25 (IBM Corp., 2 RNA could be detected in the baseline specimens prior to
Armonk, NY, USA). performing the 1% hydrogen peroxide mouthrinse. Therefore,
these two patients were excluded from the study.
Table 2 shows the viral load of the remaining 10 individual
Results patients at baseline and 30 min after the 1% hydrogen peroxide
mouthrinse. The baseline specimens exhibited a median (1st; 3rd
Patient population quartile) viral load of 1.8 × 103 (3.1 × 102; 4.7 × 104) copies/mL
of SARS-CoV-2 RNA. The second analysis 30 min after the 1%
Twelve SARS-CoV-2-positive patients were included in this hydrogen peroxide mouthrinse showed a median (1st; 3rd quar-
study. These 12 patients (6 female and 6 male) had a median tile) viral load of 1.5 × 103 (8.3 × 102; 3.4 × 104) copies/mL of
age of 55 years (range: 22–81 years). One patient was SARS-CoV-2 RNA (Fig. 2). There were no significant

Table 1 Patient characteristics

Patient no. Sex Age Admission symptoms Underlying diseases Period between diagnosis
and study inclusion (days)

1* w 60 Reduced general condition, cough, fever Obesity, arterial hypertension 5

2 w 55 Fever, genitourinary infection H/O liver transplant with 3
immunosuppression
3 m 56 Loss of appetite Cirrhosis of the liver 2
4 m 43 Reduced general condition, dyspnea Granulomatosis with polyangiitis, 1
H/O renal cell carcinoma
5 m 77 Cough, dyspnea Chronic lymphocytic leukemia, multiple 1
myeloma, arterial hypertension
6 w 57 Hydropic decompensation Cirrhosis of the liver, coronary heart disease, 3
depression, restless legs syndrome
7* w 81 Tiredness Anemia, H/O aortic valve replacement 4
8 w 47 Reduced general condition, cephalalgia, Epilepsy 1
sore throat
9 m 22 Reduced general condition, dyspnea No underlying diseases 5
10 m 67 No symptoms. Positive in Symptomatic coronary heart disease 4
preoperative screening
11 w 39 Acute on chronic renal failure, exsiccosis Chronic renal failure caused by familial 3
primary hypomagnesemia with
hypercalciuria and nephrocalcinosis
12 m 61 Reduced general condition, cough, fever Chronic renal failure, arterial hypertension, 5
coronary heart disease, obstructive sleep
apnea, obesity

*These two patients were excluded from this study since no SARS-CoV-2 RNA could be detected in the baseline specimens prior to performing the 1%
hydrogen peroxide mouthrinse
3710 Clin Oral Invest (2020) 24:3707–3713

Fig. 1 Flow of patients through the stages of this study

differences between baseline viral load and viral load 30 min copies/mL of SARS-CoV-2 RNA at baseline. Replicating vi-
after the 1% hydrogen peroxide mouthrinse (p = 0.96). rus could only be detected in one baseline specimen (Table 2).

Virus culture
Discussion
Virus culture was performed by infecting Vero cells and
assessing the viral load in the supernatants 7 days post- The COVID-19 pandemic caused an exceptional situation
infection for the specimens that exhibited more than 103 where HCPs needed to develop infection control regimens

Table 2 Intraoral viral load and

virus culture Patient no. Baseline 30 min after gargling with 1% hydrogen
peroxide mouthrinse

Copies/mL of Virus culture Copies/mL of Virus culture

SARS-CoV-2 RNA SARS-CoV-2 RNA

1* 0 / / /
2 5.7 × 102 / 6.3 × 102 /
3 7.9 × 102 / 2.9 × 102 /
4 2.9 × 102 / 1.2 × 103 /
5 9.6 × 104 Negative 1.0 × 105 Negative
6 2.8 × 103 Negative 1.1 × 103 Negative
7* 0 / / /
8 4.8 × 106 Negative 9.3 × 104 Negative
9 3.2 × 102 / 8.9 × 102 /
10 3.7 × 103 Negative 1.4 × 104 Negative
11 2.9 × 102 / 1.7 × 103 /
12 3.0 × 104 Positive 3.7 × 103 Negative

*These two patients were excluded from this study since no SARS-CoV-2 RNA could be detected in the baseline
specimens prior to performing the 1% hydrogen peroxide mouthrinse
Solidus (/), not performed
Clin Oral Invest (2020) 24:3707–3713 3711

Fig. 2 Copies/mL of SARS-

CoV-2 RNA at baseline and
30 min after 1% hydrogen perox-
ide mouthrinse. All results are
depicted as boxplots, which dis-
play medians, 1st and 3rd quar-
tiles (box), minima and maxima
(whiskers)

and treatment strategies with very little scientifically support- This study revealed that a 1% hydrogen peroxide mouthrinse
ed data at hand, which led to clinical implementation of new had no effect on reducing the intraoral viral load in SARS-CoV-
concepts in a rapidity which was hardly ever seen in medical 2-positive subjects. Therefore, any mechanical effects due to the
history [31]. In the fields of dentistry and maxillofacial sur- mouthrinse itself irrespective of its ingredients can be excluded.
gery, preprocedural mouthrinses with hydrogen peroxide As RT-PCR-based analysis is only able to detect RNA copies but
were recommended by American and German specialist soci- cannot give any indication on the infectivity of the detected virus
eties in March and April 2020 for potentially reducing the fragments, we further tried to culture SARS-CoV-2 virus from
intraoral viral SARS-CoV-2 burden before performing the patients whose baseline specimens exhibited a viral load of at
intraoral procedures [19, 20]. Interestingly, this recommenda- least 103 RNA copies per mL. These specimens were used for
tion was promptly implemented in clinical practice [12, 17, infecting Vero cells, and the viral load was determined in the
25], despite lack of any clinical data [23, 24]. On the contrary, supernatants 7 days post-infection. Here, active virus replication
there was just data from one in vitro study showing that a could only be detected from one baseline specimen, thus not
product containing 0.5% “accelerated” hydrogen peroxide allowing conclusions on the effects of hydrogen peroxide on
(Virox® Technologies Inc., Oakville Canada) was able to SARS-CoV-2 infectivity. Wölfel et al. showed that virus culture
readily inactivate coronaviruses on inanimate surfaces within depends on viral load with specimens containing less than 106
1 min [21, 22, 26, 32]. Accordingly, the rapid development of RNA copies per mL hardly yielding successful culture [36].
clinical trials on the efficacy of suchlike preprocedural Therefore, the rather low median viral load of 1.8 × 103 RNA
mouthrinses was strongly encouraged in a recent literature copies/mL in the baseline specimens may be accounted for the
review [23]. Therefore, this study aimed to investigate the absence of active virus replication after infection of cell cultures
effects of a 1% hydrogen peroxide mouthrinse on the intraoral in most specimens of the present study. Likewise, To et al. found
viral load in SARS-CoV-2-positive subjects. active virus in only 3 out of 12 saliva specimens from SARS-
This pilot study comprised 10 SARS-CoV-2-positive sub- CoV-2-positive patients despite that the median viral load in
jects, who served as their own controls. Therefore, no addi- these specimens was markedly higher (3.3 × 10 6 RNA
tional control group (e.g., using a placebo mouthrinse without copies/mL) than in the present study [35]. The rather low median
hydrogen peroxide) was deemed necessary for this pilot study. numbers of RNA copies per mL found in this study may mainly
The intraoral viral load of each patient was examined at base- be due to the exclusion of patients with indication for intubation
line and after gargling with 20 mL 1% hydrogen peroxide for or mechanical ventilation, because it is well-known that the oro-
30 s. For sampling, oropharyngeal specimens collected by pharyngeal and nasopharyngeal SARS-CoV-2 viral load strong-
mouth and throat rinses were chosen because recent studies ly correlates with COVID-19 severity [24, 37]. On the other
showed that these specimens contain a higher viral load than hand, inclusion of hospitalized patients with rather mild symp-
nasopharyngeal or oropharyngeal swabs [33], and thus may toms of COVID-19 like in the present study may rather represent
also improve accuracy of SARS-CoV-2 detection [34]. The the patient population of interest for the scope of this study.
patients themselves performed the sampling under the super- Asymptomatic patients or patients with mild symptoms, who
vision of a HCP without any need of invasive procedures, thus are not aware that they are SARS-CoV-2 positive, may tend to
reducing the risk of nosocomial SARS-CoV-2 transmission to visit dental, maxillofacial, or otorhinolaryngological practices
HCPs [34, 35]. more often than those with severe symptoms.
3712 Clin Oral Invest (2020) 24:3707–3713

Despite the small number of patients included in this pilot Ethical approval All procedures performed in this study were in accor-
dance with the ethical standards of the institutional research committee
study, the results reported here clearly show that gargling mouth
and with the 1964 Helsinki declaration and its later amendments. The
and throat with a 1% hydrogen peroxide mouthrinse for 30 s study design was approved by the ethics committee of the University of
does not decrease the intraoral viral load in SARS-CoV-2- Regensburg (ref. 20-1787-101). The study has been registered at the
positive subjects. In a very recent in vitro study, Bidra et al. German Clinical Trials Register (ref. DRKS00022484).
investigated the virucidal effects of 1.5% and 3.0% hydrogen
Informed consent Written informed consent was obtained from all in-
peroxide and 1.0%, 2.5% and 3.0% povidone iodine toward dividual patients included in this study.
SARS-CoV-2 [38]. They incubated the test compounds with
virus solution for 15 or 30 s, respectively, and then conducted a Open Access This article is licensed under a Creative Commons
standard end-point dilution assay by plating the serially diluted Attribution 4.0 International License, which permits use, sharing, adap-
tation, distribution and reproduction in any medium or format, as long as
surviving virus on Vero 76 cells. Presence or absence of cyto- you give appropriate credit to the original author(s) and the source, pro-
pathic effects was assessed after incubation for 5 days. They vide a link to the Creative Commons licence, and indicate if changes were
found that 1.5% and 3.0% hydrogen peroxide had only minimal made. The images or other third party material in this article are included
virucidal activity toward SARS-CoV-2, whereas povidone io- in the article's Creative Commons licence, unless indicated otherwise in a
credit line to the material. If material is not included in the article's
dine led to the complete inactivation of SARS-CoV-2 at the Creative Commons licence and your intended use is not permitted by
lowest concentration of 0.5% and the lowest contact time of statutory regulation or exceeds the permitted use, you will need to obtain
15 s [38]. Based on this in vitro data, further studies should permission directly from the copyright holder. To view a copy of this
investigate the effects of povidone iodine mouthrinses on the licence, visit http://creativecommons.org/licenses/by/4.0/.
intraoral viral load in SARS-CoV-2-positive subjects [24].
In view of the results of this clinical pilot study and the
in vitro results reported by Bidra et al. [38], the recommendation
of a preprocedural mouthrinse with hydrogen peroxide before
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