Disinfection of otorhinolaryngological endoscopes with electrolyzed acid water: A cross-sectional and multicenter study.

Glutaraldehyde, a germicide for reprocessing endoscopes that is important for hygiene in the clinic, might be hazardous to humans. Electrolyzed acid water (EAW) has a broad anti-microbial spectrum and safety profile and might be a glutaraldehyde alternative. We sought to assess EAW disinfection of flexible endoscopes in clinical otorhinolaryngological settings and its in vitro inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and bacteria commonly isolated in otorhinolaryngology. Ninety endoscopes were tested for bacterial contamination before and after endoscope disinfection with EAW. The species and strains of bacteria were studied. The in vitro inactivation of bacteria and SARS-CoV-2 by EAW was investigated to determine the efficacy of endoscope disinfection. More than 20 colony-forming units of bacteria at one or more sampling sites were detected in 75/90 microbiological cultures of samples from clinically used endoscopes (83.3%). The most common genus detected was Staphylococcus followed by Cutibacterium and Corynebacterium at all sites including the ears, noses, and throats. In the in vitro study, more than 107 CFU/mL of all bacterial species examined were reduced to below the detection limit (<10 CFU/mL) within 30 s after contact with EAW. When SARS-CoV-2 was treated with a 99-fold volume of EAW, the initial viral titer (> 105 PFU) was decreased to less than 5 PFU. Effective inactivation of SARS-CoV-2 was also observed with a 19:1 ratio of EAW to the virus. EAW effectively reprocessed flexible endoscopes contributing to infection control in medical institutions in the era of the coronavirus disease 2019 pandemic.

Introduction

Flexible endoscopes, which are used in a wide range of clinical specialties, including otorhinolaryngology and gastroenterology, were initially used for diagnostic purposes only; however, they have recently been increasingly used in clinical practice for screening, diagnosis, and treatments. This has resulted in the increased exposure of patients to endoscopic examinations. Compared to gastrointestinal endoscopy or bronchoscopy, flexible endoscopic examinations in the clinical practice of otorhinolaryngology and head and neck surgery have the following characteristics. First, endoscopic examinations are performed without sedation in a wide range of outpatient departments, including clinics, general hospitals, and tertiary centers. Second, they are often performed without an appointment on the day of the visit, making it difficult to predict the number of endoscopic examinations per day. Finally, the number of endoscopic examinations per day in a single facility is much larger than that of endoscopes owned by that facility. In the field of otorhinolaryngology, endoscopes have direct contact with bodily fluids or mucus containing infectious microorganisms such as otorrhea, nasal discharge, saliva, and sputum, and thus are associated with the risk of contamination. Therefore, knowledge of and techniques related to infection control are essential when using flexible endoscopes in otorhinolaryngology. Indeed, cases of gastrointestinal endoscopy-transmitted accidental infections have been reported since the 1970s in the United States (US) [1, 2], and cases of endoscopy-transmitted infections, including Helicobacter pylori, have been reported in Japan [3]. More recently, outbreaks of multidrug-resistant bacterial infections, including duodenal endoscopy-transmitted carbapenem-resistant Enterobacteriaceae infection, have been reported in the US [4]. As the demands for hygiene increases, endoscopes should be adequately and efficiently reprocessed to prevent endoscope-transmitted infections. To achieve this, it is necessary to comply with the guidelines and/or manuals on endoscope reprocessing suitable for each specialty, such as gastroenterology [5-8] or otorhinolaryngology [9, 10]. Furthermore, with the increasing demand for disinfection and the prevalence of endoscopy in the field of otorhinolaryngology, a low-cost and safe disinfection method is eagerly awaited for endoscopic reprocessing. This is particularly true because of the emergence of COVID-19 in 2019, the potential risk of transmission through endoscopes used in the nose or throat, and multidrug-resistant bacteria.

Glutaraldehyde, o-phthalaldehyde, and peracetic acid are commonly used high-level disinfectants; however, these chemicals are sometimes hazardous to patients and healthcare professionals [11, 12]. Therefore, ventilation during the endoscopic examination and reprocessing should be considered. A popular alternative to central decontamination is the chlorine dioxides wipe system. Chemical decontamination utilizing wipe systems, such as chlorine dioxide, is acceptable if an endoscopic washer disinfector is unavailable. The chlorine dioxide system is much less expensive but deemed an inferior method of decontamination. The system requires staff to be thoroughly trained and conversant with the technique and introduces the risk of human error [9]. In contrast, electrolyzed acid water (EAW) contains a mixture of oxidizing species. An EAW is generated at the anode of a dual-chamber electrolytic cell with a membrane placed between the anode and cathode by electrolyzing a low-concentration aqueous solution of sodium chloride. At the anode, chloride ions are converted into gaseous chlorine, which then reacts with water to form hypochlorous acid, which plays a major role in disinfection, and results in the formation of EAW at pH 2.7. EAW has a broad antibacterial spectrum and has been shown to remove spore-forming bacteria [13], acid-fast bacteria [14, 15], fungi [16, 17], and blood-borne infection-causing viruses such as hepatitis B virus [18, 19], and human immunodeficiency virus [20]. EAW has also been experimentally proven to be minimally cytotoxic [16]. It is essential to use EAW properly for reprocessing endoscopes because it is corrosive to metals when the chlorine concentration is high, and it does not provide a sufficient disinfection effect when the chlorine concentration is low. Tsuji et al. demonstrated that EAW eliminates various bacteria and viruses within 5 min in upper gastrointestinal endoscopes, which have a high rate of contamination with bacteria including Helicobacter pylori. They also showed the potential of using EAW to solve several clinical problems, such as the toxicity of liquid chemical germicides, prolonged exposure to endoscope disinfection, and the high costs associated with the use of other types of high-level disinfectants [16]. Although EAW has been used to reprocess gastrointestinal endoscopes for more than 20 years and is effective at disinfecting clinically used gastrointestinal endoscopes [16], its efficacy in disinfecting clinically used otorhinolaryngological endoscopes has not been reported.

In the present study, we evaluated the efficacy of EAW for the disinfection of otorhinolaryngological endoscopes in clinical settings. We further conducted a microbiological analysis of the surface of endoscopes, and verified the efficacy of EAW in removing possible pathogenic bacteria related to otorhinolaryngology [21] or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by an in vitro study.

Materials and methods

Clinical study

The study was conducted as a multicenter research trial with 90 patients who underwent flexible endoscopic examinations in the Department of Otorhinolaryngology at three medical sites: a primary care clinic, a secondary care hospital, and a tertiary care center (30 patients per site).

Microbiological tests

First, to verify contamination, samples were collected before the manual cleaning of used otorhinolaryngological endoscopes. For non-channeled endoscopes, samples were collected at two sites: the overall area of the flexible tube and bending tip inserted into the patient (insertion site) (A-1) and the overall area of the control handle and angulation knob operated by the examiner (operation site) (A-2). For channeled endoscopes with a suction/forceps channel, samples were collected at three sites, including A-1, A-2, and the inside of the operating channel (A-3) (Fig 1). A sample was collected at A-1 and A-2 by swabbing the sampling site with a wet bacteriological swab (ST25-100, ELMEX Co., Ltd., Tokyo, Japan) ten times, and the swab was stored in a sterile tube. At A-3, the suction/forceps channel was flushed with 20 mL of sterile normal saline from the forceps channel inlet, and the saline was collected in a sterile tube at the forceps channel outlet and stored. Subsequently, the endoscopes were cleaned in accordance with the recommendation on reprocessing techniques of flexible otorhinolaryngological endoscopes [10] as follows:

Fig 1

Sites of microbiological sampling in otorhinolaryngological endoscopes.

Microbiological samples were collected at two or three sites for non-channeled endoscopes or channeled endoscopes with a suction/forceps channel, respectively, before (A-1, 2, 3) and after (B-1, 2, 3) cleaning and disinfection with EAW using a Cleantop KD-1. A-1 and B-1: overall area of the flexible tube and bending tip inserted into the patient (insertion site); A-2 and B-2: overall area of the control handle and angulation knob operated by the examiner (operation site); A-3 and B-3: inside of the operating channel.

After sampling, the outside surface of the endoscope was wiped with Tanpaclean wipes (Kaigen Pharma Co., Ltd., Osaka, Japan) and gauze was soaked in a cleaning solution.

The outside surface of the endoscope was cleaned with a sponge and detergent containing protease enzymes, followed by rinsing with water to remove residual blood, body fluids, and proteins.

For channeled endoscopes with a suction/forceps channels, the inside of the channel was cleaned with a brush and flushed with at least 200 mL of cleaning solution to remove contaminants.

The enzyme detergent was thoroughly removed by washing with tap water, and the inside of the channel was flushed with water to wash out the detergent.

After manual cleaning, the endoscopes were disinfected using a Cleantop KD-1 (Kaigen Pharma Co., Ltd.), a disinfector designed to disinfect flexible endoscopes with EAW. The effective chlorine concentration of Cleantop KD-1 was between 10 and 40 ppm, and a course of disinfection took 90 s. During the cleaning and disinfection of the endoscopes, personal protective equipment such as masks, gloves, goggles, and gowns were worn appropriately, and adequate ventilation was provided in the room.

Immediately after cleaning and disinfection, contaminated samples were collected from the endoscopes in the same manner as before cleaning and disinfection. For non-channeled endoscopes, samples were collected at two sites: the overall area of the flexible tube and bending tip inserted into the patient (insertion site) (B-1), and the overall area of the control handle and angulation knob operated by the examiner (operation site) (B-2). For channeled endoscopes, samples were collected at three sites: B-1, B-2, and inside the operating channel (B-3) (Fig 1).

All samples collected were promptly cultured for aerobic bacteria, anaerobic bacteria, and fungi at the Japan Microbiological Clinic Co., Ltd. (Kanagawa, Japan), followed by bacterial culture to calculate the proportion of effectively disinfected endoscopes and identify species and strains of bacteria present. The analysis included the endoscopes used in patients with more than 20 colony-forming units (CFU) of bacteria at one or more sampling sites before disinfection. Effective disinfection was defined as a bacterial count of ≤20 CFU per sampling site and no detection of indicator microorganisms after disinfection based on the European Society of Gastrointestinal Endoscopy and European Society of Gastroenterology Nurses and Associates Guidelines for Quality Assurance in Reprocessing: Microbiological Surveillance Testing in Endoscopy [22].

In vitro studies for the antibacterial and antiviral response of EAW

A dual-chamber electrolytic cell was equipped with platinum-coated titanium electrodes (11 × 19 cm each, CT-501S, Tanaka Kikinzoku Kogyo K. K., Tokyo, Japan) with the chambers separated by a cationic membrane (Nafion 424, DuPont, DE, USA) used to produce EAW. EAW was obtained at the anode side by electrolyzing 500 mL of 0.1% sodium chloride solution for 6–7 min at a constant voltage of 24 V. The free (available) chlorine concentration was measured using a chlorine meter (DP-3F, Kasahara Chemical Instruments Corp., Saitama, Japan), and the pH was measured using a pH meter (HM-31P, DKK-TOA Corp., Tokyo, Japan). The effective chlorine concentration, pH, and oxidation-reduction potential of EAW used in bacteria experiments were adjusted to 20.66 ± 0.18 ppm, 2.60 ± 0.00, and 1133.7 ± 3 mV, respectively. For SARS-CoV-2 experiments, they were adjusted to 10.57 ± 0.34 ppm, 2.61 ± 0.01, and 1119.5 ± 6.2 mV, respectively.

Bacterial culture and bactericidal effects of EAW

The bacteria used in the tests were Staphylococcus aureus NBRC13276, Streptococcus pneumoniae NBRC102642, Streptococcus pyogenes GTC262, Klebsiella pneumoniae NBRC3512, Haemophilus influenzae IID983, and clinical isolates of β-lactamase-negative ampicillin-resistant Haemophilus influenzae (BLNAR) nos.1 and no.2. S. pyogenes GTC262 was provided by the Center for Conservation of Microbial Genetic Resources, Gifu University, through the National Bioresource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. H. influenzae IID983 was provided by the Institute of Medical Science, The University of Tokyo, through the NBRP of MEXT, Japan. S. pyogenes was cultured on blood agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) in an anaerobic environment. S. pneumoniae was cultured on blood agar (Nissui Pharmaceutical Co., Ltd.), S. aureus and K. pneumoniae were cultured on heart infusion agar (Eiken Chemical Co., Ltd., Tokyo, Japan), and H. influenzae was cultured on chocolate agar (Nissui Pharmaceutical Co., Ltd.) in an aerobic environment. Bacterial cells were cultured on plates for 16 h and collected. To prepare bacterial suspensions for testing, the bacterial cells were collected and suspended in distilled water to adjust a concentration same as the turbidity of McFarland Standard No. 2. Subsequently, EAW and the bacterial suspension were mixed in a 1:1 ratio. After they were in contact for a designated period, free chlorine was neutralized with an equal volume of 1% bovine serum albumin solution [23]. To quantify residual viable bacterial cells, a specimen neutralized for a designated period was serially diluted and spread on plates. The plates were cultured overnight at 37°C, and the number of colonies was determined to calculate the number of residual viable bacterial cells.

EAW treatment of SARS-CoV-2

Two isolates of SARS-CoV-2 (WK-521 [provided by the National Institute of Infectious Diseases, Tokyo, Japan] and OMC-510) were amplified and titrated using Vero E6/TMPRSS2 cells as described previously [24]. Infectious titers of virus stocks of WK-521 and OMC-510 were 1.2 × 107 plaque-forming units (PFU)/mL and 5.0 × 107 PFU/mL. EAW was added to each virus stock at a 990 μL:10 μL or 950 μL:50 μL ratio, and reacted at room temperature for 1 min. Subsequently, 100 μL of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum was added to the mixture to neutralize the EAW and further incubated at room temperature for 5 min. After incubation, the viral titer in the treated samples were determined as PFU/mL. Sodium chloride solution (0.1%) was used as a control.

Ethical approval

This study was performed in accordance with the ethical standards of the Declaration of Helsinki of 1975 and its later amendments or comparable ethical standards. The study protocol of the present study was approved by the Institutional Review Boards and Ethics Committees of Kyoto University Hospital and Kyoto University Graduate School of Medicine (R2386). Verbal informed consent for participation was obtained from patients visiting the Department of Otorhinolaryngology outpatient facility using an opt-out methodology because this study does not harm the patients in any way, including direct microbiological or blood sampling.

Results

General characteristics of patients and endoscopes

The patients included in the present study had a mean age of 59 years with a range from 1 to 92 years, and consisted of 50 males and 40 females. Of those with primary diseases that required endoscopic examination, 55 patients had laryngopharyngeal disease (pharyngitis in 9, laryngeal cancer in 4, dysphagia in 3, tongue cancer in 3, hypopharyngeal cancer in 3, oropharyngeal cancer in 3, nasopharyngeal cancer in 2, and 28 with other), 29 patients had nasal disease (sinusitis in 20, epistaxis in 5, and other condition in 4), and 6 patients had ear disease (otitis media in 4, otitis externa in 1, and ear fullness caused by sinusitis in 1) (Table 1). Regarding the non-channeled endoscopes used, Pentax VNL-90s was used in 30 patients, Olympus ENF-VH in 25, Olympus ENF-V3 in 16, Olympus ENF TYPE VQ in 8, and Olympus ENF TYPE V2 in 1. Channeled endoscopes used in this study were the Olympus ENF TYPE VT2 in 6 patients and Olympus ENF-VT3 in 4 patients.

Table 1

Primary diseases requiring endoscopic examination.

Examination sites	Identified bacterial species	Primary diseases			Total
		Tertiary care center	Secondary care hospital	Primary care clinic
Throat in 55 patients	pharyngitis	1	-	8	9
	laryngeal cancer	2	2	-	4
	dysphagia	1	2	-	3
	tongue cancer	3	-	-	3
	hypopharyngeal cancer	3	-	-	3
	oropharyngeal cancer	3	-	-	3
	nasopharyngeal cancer	2	-	-	2
	Other	2	1	-	3
Nose in 29 patients	sinusitis	-	6	14	20
	epistaxis	-	5	-	5
	Other	1	2	1	4
Ear in 6 patients	otitis media	-	2	2	4
	otitis externa	-	-	1	1
	ear fullness caused by sinusitis	-	1	-	1

Endoscope disinfection using EAW in clinical settings

After the microbiological culture of clinically used endoscopes, more than 20 CFU of bacteria at one or more sampling sites was detected in 75 of 90 patients (83.3%) (Table 2). More specifically, microorganisms were detected directly after clinical use without disinfection in 13 of 90 patients (14.4%), 74 of 90 patients (82.2%), and 9 of 10 patients (90.0%) at the endoscopic operation site, endoscopic insertion site, and inside the endoscopic suction/forceps channel, respectively. After cleaning and disinfection using EAW, microorganisms were detected in only one of 75 endoscopes inside the endoscopic suction/forceps channel with a value of 30 CFU, which represents a > ×105 reduction in a clinical environment. After reprocessing with the EAW, the remaining 74 samples showed no further bacterial contamination.

Table 2

Characteristics of patients and endoscopes included in this study.

Study site name	Number of patients	Number of endoscopes used in patients and included in analysis a	Number of endoscopes used in patients with effective disinfection b	Effective disinfection rate (%) c	Sampling site	Number of samples	Detection of microorganisms d
							Before disinfection	After disinfection
Tertiary care center	30	28	28	100	Operation site	30	10	0
					Insertion site	30	28	0
					Inside the suction/forceps channel	10	10	0
Secondary care hospital	30	25	24	96	Operation site	30	6	0
					Insertion site	30	25	0
					Inside the suction/forceps channel	1	1	1
Primary care clinic	30	22	22	100	Operation site	30	5	0
					Insertion site	30	23	0
					Inside the suction/forceps channel	0	0	0
Total	90	75	74	98.7

a Endoscopes used in patients with more than 20 CFU of bacteria at one or more sampling sites before disinfection.

b Endoscopes with a bacterial count ≤20 CFU per sampling site and no detection of indicator microorganisms after disinfection.

c Effective disinfection rate (%) = 100 × (number of endoscopes with effective disinfection/number of endoscopes included in the analysis).

d Endoscopes with more than 1 CFU of bacteria at a sampling site.

CFU: colony-forming units.

Staphylococcus species are the most common potential pathogen in otorhinolaryngological endoscopes

The most common genus of strains detected was Staphylococcus, followed by Cutibacterium and Corynebacterium at all sites including the ear, nose, and throat (Table 3). By test location, Staphylococcus and Cutibacterium were most commonly detected at all test locations, with no differences among primary, secondary, and tertiary medical institutes. In endoscopes with ineffective disinfection, S. aureus was detected before and after cleaning and disinfection. No fungi were detected in this study.

Table 3

Bacterial species and location of isolation.

Examination sites	Identified bacterial species	Number of strains detected a			Total
		Tertiary care center	Secondary care hospital	Primary care clinic
Throat in 55 patients	Staphylococcus	40	15	12	67
	Cutibacterium	21	9	3	33
	Corynebacterium	6	2	2	10
	Streptococcus	6	1	-	7
	Pseudomonas	4	-	-	4
	Bacillus	2	1	-	3
	Rothia	2	1	-	3
	Serratia	2	1	-	3
	Klebsiella	1	-	1	2
	Other	6	2	1	9
Nose in 29 patients	Staphylococcus	2	9	12	23
	Cutibacterium	-	4	4	8
	Corynebacterium	1	5	2	8
	Bacillus	1	-	1	2
	Citrobacter	-	1	-	1
	Klebsiella	-	1	-	1
	Proteus	-		1	1
Ear in 6 patients	Staphylococcus	-	3	2	5
	Cutibacterium	-	2	1	3
	Bacillus	-	-	1	1

a Multiple strain were detected in some samples.

EAW has a wide range of in vitro activity as a germicide against various microorganisms

In the in vitro study, EAW was added to each bacterial suspension in McFarland No. 2 standard, and viable counts were determined at each specific time point to determine the bactericidal activity of EAW (Table 4). To determine the relationship between drug resistance and disinfection resistance, a similar test was performed using clinical isolates of BLNAR. EAW reduced the viable counts of all bacteria to <107 within 30 s and successfully removed drug-resistant bacteria. No controls were used in this study, however, we confirmed that microorganisms not treated with EAW survived at more than 107 under the same culture conditions.

Table 4

Reduction of bacteria by EAW.

Bacterial strain	Initial density (CFU/mL)	After EAW contact (30 s)
Streptococcus pneumoniae NBRC102642	1.1 × 107	n.d.
Streptococcus pyogenes GTC262	7.9 × 107	n.d.
Staphylococcus aureus NBRC13276	3.8 × 107	n.d.
Klebsiella pneumoniae NBRC3512	9.8 × 107	n.d.
Haemophilus influenzae IID983	2.9 × 107	n.d.
Clinical isolates of β-lactamase-negative ampicillin-resistant Haemophilus influenzae (BLNAR) no.1	3.9 × 107	n.d.
Clinical isolates of β-lactamase-negative ampicillin-resistant Haemophilus influenzae (BLNAR) no. 2	1.0 × 108	n.d.

n.d.: below the limit of detection (<10 CFU/mL); EAW: electrolyzed acid water; CFU: colony-forming units; BLNAR: β-lactamase-negative ampicillin-resistant Haemophilus influenzae.

EAW has inactivating activity against SARS-CoV-2

After contacting the SARS-CoV-2 suspensions for 1 min, EAW inactivated SARS-CoV-2 by ≥99.9% in all virus suspensions at different mixture ratios (Table 5). Furthermore, a mixture ratio of 99:1 was associated with a greater reduction in viral load than a ratio of 19:1 and ≥99.9% inactivation.

Table 5

Inactivating activity of EAW against SARS-CoV-2.

Isolates	Titer of virus stock a	Mixing ratio virus: EAW	Titer of EAW-contacted virus a	Titer of control-contacted virus a
SARS-CoV-2/WK-521	1.2 × 107	1:19	50	7.0 × 105
		1:99	n.d.†	1.3 × 105
SARS-CoV-2/OMC-510	5.0 × 107	1:19	2.2 × 103	3.4 × 106
		1:99	n.d.	5.0 × 105

a PFU/mL. n.d.: below the limit of detection (< 5 PFU/mL); EAW: electrolyzed acid water; PFU: plaque-forming units; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Discussion

In this study, microorganisms were detected once in a channeled endoscope used in 90 patients after disinfection of the inside of the channel. Several factors may have limited the efficacy of the disinfection procedures. Potential causes included (1) contamination by airborne bacteria in the environment or normal skin flora of a sampler during sampling, (2) inadequate cleaning and disinfection of the endoscope in a washer disinfector, and (3) the effect of biofilms. Contamination, as described in (1), cannot be completely ruled out for the test system in this study. Inadequate cleaning and disinfection as described in (2) should be ruled out because EAW is sufficiently effective enough to eliminate S. aureus, the species detected after cleaning and disinfection. The effect of biofilms, as described in (3), cannot be ruled out as a possible cause of residual bacteria after disinfection with EAW. These findings indicate the importance of properly storing endoscopes after cleaning and disinfection. The channeled endoscope in which residual bacteria were detected had been stored in a carrying case for more than a month after its last use rather than in a storage cabinet. Because channeled endoscopes with a suction/forceps channels might be used infrequently in community hospitals and clinics in Japan, they might remain unused for more than a month after their last use, which was the case in the present study. Carrying cases for endoscopes are not sufficiently ventilated and provide conditions favorable for pathogen growth. Particular caution should be exercised when storing a channeled endoscope, because water tends to remain in the channel. The guidance states that no endoscope should be stored in a coiled position nor in a carrying case [10].

The present clinical study demonstrated that EAW inactivated the gram-positive bacteria S. aureus, S. pyogenes, and S. pneumoniae and the gram-negative bacteria H. influenzae and K. pneumoniae, which cause otorhinolaryngological diseases [21], although these strains were not often detected. Our in vitro data also showed that EAW exhibited a wide range of microbicidal activities against bacteria frequently isolated from otorhinolaryngological sites. No fungi were detected in the present study; however, Urata et al. demonstrated that EAW has microbicidal activity against Candida albicans, one of the major fungal species isolated from the oral cavity or throat [17]. These findings suggest the suitability of EAW for disinfection of endoscopes in a otorhinolaryngological environment.

In our in vitro study on the inactivation of SARS-CoV-2, EAW inactivated SARS-CoV-2 by ≥99.9% within 1 min at an effective chlorine concentration of 10 ppm, and a mixture ratio of 99:1 was associated with greater inactivation than a ratio of 19:1. This suggests that the protein content in the virus suspensions affected the inactivation activity of EAW. Because more than 5 L of EAW is used to disinfect a flexible endoscope in clinical settings, a mixture ratio of 99:1 may more closely mimic actual use in clinical settings. In addition, the thorough removal of contaminants, such as proteins, by conventional preliminary manual cleaning is important to ensure the consistent disinfection performance of EAW in clinical settings. EAW has been used to disinfect SARS-CoV-2 as an alternative to alcohol. Takeda et al. reported that the viricidal activity of EAW against SARS-CoV-2 depended on the amount of free available chlorine, indicating that an acidic solution without free available chlorine does not inactivate SARS-CoV-2 over a short period [25]. These findings were supported by an in vitro study by Xiling et al., which showed that free available chlorine at 1,000 mg/L inactivated SARS-CoV-2 in 30 s [26].

Regarding the limitations of this clinical study, we designed it to detect aerobic bacteria, anaerobic bacteria, and fungi, however, it is possible that not all microorganisms could be detected. Considering this, it is sufficient to confirm the trend of microorganisms detected in the field of otorhinolaryngology and the disinfection effect of EAW. Taken together, EAW is a safe, broad-spectrum disinfectant that can be used for reprocessing endoscopes during the coronavirus disease 2019 pandemic.

Conclusion

This study demonstrated that the EAW system for reprocessing flexible endoscopes appears to be an ideal disinfection system with a broad anti-microbial spectrum with both bactericidal and viricidal effects and safety profiles. EAW does not harm human tissues, therefore, it could be an alternative to commonly used high-level disinfectants including glutaraldehyde, o-phthalaldehyde, and peracetic acid. Because EAW disinfectant production requires only salt, tap water, and electricity, and EAW loses its oxidative and acidic properties when exposed to the environment, the use of EAW in the disinfection of otorhinolaryngological endoscopes should contribute to infection control in medical institutions from the viewpoints of safety for medical staffs and environmental friendliness in order to overcome supply crisis during the coronavirus disease 2019 pandemic, indicating sustainable systems and enabling safe continuity.

16 Aug 2022

9 Sep 2022

PONE-D-22-18329

Disinfection of otorhinolaryngological endoscopes with electrolyzed acid water: a cross-sectional and multicenter study

PLOS ONE

Dear Dr. Awatif Abid Al-Judaibi,

Academic Editor

PLOS ONE

We would like to thank you and the Reviewers for the thought-provoking comments on our manuscript. We have revised the paper in accordance with the comments made by the Reviewers, and made our point-by-point responses as follows.

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＞ Thank you so much for your comment. We have read through two of the PLOS ONE style templates and confirm that our manuscript meets PLOS ONE's style requirements.

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"I have read the journal's policy and the authors of this manuscript have the following competing interests: Protein Clean Sheets and a CLEANTOP KD-1 automatic washer and disinfector used in the present study were provided by Kaigen Pharma, Co., Ltd. and one of the authors, Hideki Hayashi, is an employee of Kaigen Pharma, Co., Ltd."

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＞ We have included the statement "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” in the cover letter.

3. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

> Thank you for your suggestion. We have reviewed the reference list again and confirm that it is correct.

Additional Editor Comments:

Reviewer 1:

1- Significant differences between A-1, A-2, and A-3. sampling sites should be mentioned.

> We really appreciate the reviewer's suggestion. As this comment would be related to reviewer 3's comment No. 4, we have edited the new Fig. 1 to clearly demonstrate the sampling sites.

2- There are some typos and grammatical errors which should be revised.

> Thank you for your careful reading. We have revised the manuscript again, and also used an English language editing service provided by Editage (www.editage.com).

3- This sentence is not correct "The verbal informed consent for participation was obtained form patients visiting the outpatient facility of the

Department of Otorhinolaryngology using an opt-out methodology because this study does not harm the patients in any way including direct microbiological sampling or blood sampling"

> This comment would be related to reviewer 3's comment No. 13. We fixed the typo "form" into "from". We also rewrote the sentence for clarity (Page 13, line 221 - 224).

4- I couldn't find any of the references from the 21, 22 and 23

Reference correction is necessary

> We appreciate careful review by Reviewer 1. We believe this is not the case. Reference 21 was cited in line 157, 22 was cited in line 189, 23 was cited in line 196, in the former form of manuscript. In the current form of manuscript, reference 22 (changed from previous reference 21) is cited in Page 10, line 169, reference 23 (changed from previous reference 22) is cited in Page 12, line 201, and reference 24 (changed from previous reference 23) is cited in Page 12, line 209.

Reviewer 2:

Authors have reported effective use of Electrolyte acid water (EAW) in disinfecting otorhinolaryngological endoscopes, which is of clinical significance. Authors have presented sufficient data in support of research question keeping limitation of study in account. Manuscript can be accepted for publication after making amendments as suggested.

> We really appreciate the reviewer's careful and fruitful comments.

Some specific comments/reviews to incorporate/amend are as follows:

1- Abstract: Authors have summarized key findings appropriately. However, in vitro inactivation of bacteria and SARS-CoV-2 need to be presented consistently in number/percentage or CFU to enhance reader's understanding.

> Thank you for the important comment. We rewrote the abstract and added data in number, ratio, and CFU (Page 3, line 41 - 43).

2-Introduction: Significance of using EAW for disinfection of otorhinolaryngological endoscopes for bacteria has been presented appropriately. however, authors need to include significance of using EAW for SARS-CoV-2 as well briefly.

> We really appreciate the reviewer's suggestion. We added the part describing relationship between endoscope reprocessing and SARS-CoV-2 (Page 5, line 73 - 77).

3- Methods: Authors used appropriate methodology to address the research question. However, following amendments 13are suggested:

Page 8, lines 138-139: Authors either need to include time/concentration for disinfection using Cleantop-KD-1 or reference for the procedure.

> We thank the reviewer for raising this point. We added time and concentration for disinfection with Cleantop-KD-1 (Page 9, line 150 -152).

Page 11, lines 190-191: "Overnight" incubation time need to be rephrased in terms of "Hours" for clarity.

> We also rephrased "for 16 h" instead of "overnight" for clarity (Page 12, line 197).

4- Results: Authors have presented results appropriately using tables facilitating reader's understanding. Suggested amendment:

Page 13, lines 220-221: "29 patients had nasal disease....." needs correction as breakdown adds up to 30.

> We really appreciate the reviewer's indication. As related to the Reviewer 3's comment No. 7, we rewrote the number of patients with sinusitis, and added the new table 1 for clarity (Page 14, line 240). According to the addition of new table 1, previous table 1, 2, 3, and 4 were renumbered into the new table 2, 3, 4, and 5, respectively.

Page 14, lines 234- 235: " ...only in 1 of 75 patients...." needs to be corrected as " ...only in 1 of 75 endoscopes...."

> According to the reviewer's suggestion, we changed "patients" into "endoscope" (Page 15, line 249).

5-Discussion: Overall needs to be rephrased. Few amendments suggested are:

Page 18-19, line 286- 301, "Because otorhinolaryngology ........high-level disinfection." is a repeat from introduction need to be deleted.

> Thank you so much for suggestion. This comment would be related to the reviewer 3's comment No. 10. We removed the indicated part from the section of Discussion.

Page 19-20, para 2, line 309-320: Needs rephrasing for clarity. Also, reference of table needs to be removed from discussion.

> We thank the reviewer for suggestion. We rewrote this part in the section of Discussion for clarity (Page 20, line 301 - Page 21, line 318). In addition, we removed the reference of table 3 in this part.

6- Conclusion: It is vague and needs rephrasing keeping context of study in view to highlight the significance of study.

> We appreciate the reviewer's suggestion. This comment would be related to the reviewer 3's comment No. 11. We totally rephrased the section of Conclusion for clarity according to the reviewers' suggestion (Page 23, line 349 - 358).

Reviewer 3:

1. Lines 90 - 93: Despite there is no study that presents the disinfection efficacy of EAW in clinically used otorhinolaryngological endoscopes, authors have mentioned about the effectiveness of using EAW in disinfecting clinically for gastrointestinal endoscopes. Authors should highlight their significant findings and contributions.

> Thank you so much for your constructive suggestion. We added a few sentences to emphasize the disinfection efficacy of EAW for gastrointestinal endoscopes highlighting findings shown by the previous study (Page 6, line 96 - 102).

2. Introduction can be improved by adding a few sentences about the hypochlorous acid since it is the primary antibacterial agent in the study.

> This comment might be related to the next comment. We totally agree with the comment and added the phrases to the part regarding hypochlorous acid (Page 5, line 86 - Page 6, line 91).

3. Line 95: "Fig 1. Schematic of generation of electrolysed acid water" is not necessary to be included in the manuscript, as it is well known.

> According to the reviewer's suggestion, we removed "previous Fig 1" that shows generation of EAW. Instead, we added the new Fig 1 to show the sampling sites in endoscopes (Page 8, line 141).

4. Lines 116 –148: it is helpful to provide an image (as supporting Information file) of the endoscopy instruments with two sites (A-1 to A-3, B-1 to B-3) to aid the understanding of this part.

> We really appreciate the reviewer's suggestion. This comment would be related to reviewer 1's comment No. 1. We accordingly have edited the new Fig. 1 to clearly demonstrate the sampling sites (Page 8, line 141).

5. Line 149 and line 340: this a study designed to culture aerobic and anaerobic bacteria as well as fungi but there is limited discussion about fungi being observed or detected or fungicidal effect, please explain more.

> This suggestion was very helpful for us. We added a few phrases regarding fungi in the section of Introduction (Page 6, line 92) and Results (Page 17, line 269) using reference 16 and 17. We renumbered the references cited in the later part of the new manuscript, accordingly. In addition, we added a few sentences to explain the fungicidal effect of EAW in the section of Discussion (Page 21, line 323 - 326).

6. Line 171: the subheading "bacteria and culture" is more generic while the paragraph discusses EAW treatment; please edit the subheading to reflect all the aspects of the method on bacterial culture and bactericidal effects of EAW.

> According to the reviewer's suggestion, we changed the subheading "bacteria and culture" into "bacterial culture and bactericidal effects of EAW" (Page 11, line 184).

7. Lines 216- 226: A table is preferred here given the amount of data being presented.

> We really appreciate the reviewer's indication. As this comment would be related to the Reviewer 2's comment No. 4, we rewrote the number of patients with sinusitis, and added table 1 for clarity (Page 14, line 240).

8. Line 270: What is the control sample for the data presented in the Table 3?

> Actually, we didn't set the control sample for data in new Table 4 (previous Table 3). Instead, we added the sentence to this part as follows; " No controls were used in this study, however, we confirmed that microorganisms not treated with EAW survived at more than 107 under the same culture conditions." (Page 18, line 281 - 283).

9. Full name of bacterial strains should be mentioned in the (Table 3) as mentioned in from line 172-175 have to be.

> According to the reviewer's comment, we added full name of bacterial strains in the new Table 4 (previous Table 3) as mentioned in from line 185 -188 in the current form of manuscript.

10. Lines 258 – 301: Discussion can be improved to avoid repetition with introduction section.

> Thank you so much for suggestion. This comment would be related to the reviewer 2's comment No. 5, we removed the indicated part from the section of Discussion.

11. The conclusion is supported by the results for EAW treatment of SARS-CoV-2 but not for bacteria. Therefore, this section must be improved and highlight why EAW can be an ideal disinfectant (bactericidal and virucidal effects) and use it at present as regarded to be safe and environmentally friendly to overcome supply crisis during pandemic emergency and enable the safe continuity.

> We really appreciate the reviewer's suggestion. This comment would be related to the reviewer 2's comment No. 6, we totally rephrased the section of Conclusion for clarity according to the reviewers' suggestion (Page 23, line 349 - 358).

12. Line 183: influenzae, make the last letter (e) italic as well.

> Thank you for your indication. We made the last latter (e) italic accordingly.

13. Line 210: form or from

> This comment would be related to reviewer 1's comment No. 3. We fixed the typo "form" into "from".

Submitted filename: Response to Reviewers.docx

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19 Sep 2022

Disinfection of otorhinolaryngological endoscopes with electrolyzed acid water: a cross-sectional and multicenter study

PONE-D-22-18329R1

Dear Dr. Koichi Omori,

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23 Sep 2022

PONE-D-22-18329R1

Disinfection of otorhinolaryngological endoscopes with electrolyzed acid water: a cross-sectional and multicenter study

Dear Dr. Omori:

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