key: cord-0736612-gbhgjd5o authors: Kanesaka, Izumo; Katsuse, Akiko Kanayama; Takahashi, Hiroshi; Kobayashi, Intetsu title: Evaluation of a bipolar ionisation device in inactivation of antimicrobial resistant bacteria, yeast, Aspergillus and human coronavirus date: 2022-04-19 journal: J Hosp Infect DOI: 10.1016/j.jhin.2022.04.004 sha: 1662defd6c06e1debb3e553e3a735b19d21d2662 doc_id: 736612 cord_uid: gbhgjd5o BACKGROUND: The efficacy of bipolar ionisation in the healthcare setting has yet to be proven. A major limitation of studies sponsored by industry has been assessment of efficiency within test chambers in which ozone levels are not adequately controlled. AIM: The objective of this study was to assess the effectiveness of bipolar ionisation against antimicrobial-resistant bacteria as well as fungi and human coronavirus within a controlled testing chamber designed to mitigate the effect of ozone. METHODS: Bacteria- and fungi-inoculated gauze pads, as well as human coronavirus 229E-inoculated stainless steel plates were placed within the vicinity of the AIO-2 bipolar ionizer and left at room temperature (2 h for coronavirus and 4 h for bacteria and fungi). FINDINGS: A 4 h exposure to bipolar ionisation showed a 1.23 to 4.76 log reduction corresponding to a 94.2 to >99.9% cfu/gauze reduction for Clostridioides. difficile, KPC-producing Klebsiella pneumoniae, meticillin-resistant Staphylococcus aureus and multidrug-resistant Staphylococcus aureus. Against human coronavirus, a 1.2 log TCID(50) reduction was observed after 2 h. CONCLUSION: The assessment of bipolar ionisation systems merits further investigation as an infection control measure. Since the occurrence of the SARS-CoV-2 (COVID-19) global pandemic, there has been a growing market for air purification and reduction of surface contamination using air ionisation devices [1] . The antimicrobial effectiveness of ions has long been controversial. A review by Krueger and Reed concluded that negative and positive ions inhibit growth of microorganisms [2] . A study focusing on static charges on fomitic surfaces and deposition of bacteria showed that bipolar ionisation resulted in reduction of bacterial deposition [3] . In a trial conducted in an intensive care unit, Kerr et al. found that negative air ionizers were associated with a significant decrease in acinetobacter infections as well as patient colonization [4] . In a recent real-world J o u r n a l P r e -p r o o f hospital setting, the implementation of a technology using charged particles reduced healthcare-associated infections (HAI) by 45% [5] ). Of the various technologies claiming to deactivate bacteria, viruses and fungi, variants of bipolar ionisation technology (needle-point, corona discharge, plasma cluster etc.) have enjoyed renewed commercial popularity as a result of the COVID-19 pandemic. In bipolar ionisation, positive (H+) and negative (O2-) ions are generated when water molecules are exposed to high-voltage electrodes. The mechanism(s) associated with the biocidal effect of positive and negative ions have not been clearly established. The purported mechanism of the inactivation of microorganisms and viruses is the clustering of these ions around viruses and microorganisms resulting in the formation of OH radicals, which remove hydrogen and the formation of water vapors leading to inactivation [6] . While droplet and airborne transmission are considered to be the main route of exposure in the ongoing COVID-19 pandemic, disinfection of contaminated or potentially contaminated surfaces is also one of the strategies for controlling COVID-19, as it has been shown that SARS-CoV-2 can remain viable on different surfaces from hours to a few days [7] . In this study, we performed an independent evaluation of a bipolar ionisation technology device against antimicrobial-resistant bacteria, Candida albicans, Aspergillus fumigatus, and human coronavirus under a controlled laboratory environment in which ozone as well as the concentration of ions were not allowed to accumulate in an enclosed environment. Our objective was to assess the potential utility of this technology within a healthcare environment as an adjunct to existing protocols for minimizing healthcare-associated infections. The organisms tested were ATCC strains of meticillin resistant For A. fumigatus, a spore suspension was prepared and diluted using physiological saline for a spore suspension of approximately 1 x 10 5 spores/mL. Similarly, a suspension of C. albicans equivalent to approximately 1.0 x 10 6 cfu/mL was prepared. One mL of the prepared bacterial and fungal suspensions was applied to a sterile, 5 cm x 5 cm gauze pad (Kawamoto Corporation, Osaka, Japan) and left at room temperature [8] . Sterile 1.5 cm x 1.5 cm SUS304 (Japanese SIS standard) stainless steel squares were loaded into sterile Petri dishes using sterile forceps. A 4.3 log10 TCID50/mL suspension of human coronavirus 229E tissue culture was applied to the surface of the stainless-steel squares and left at room temperature [9] . Based on several other publications on the evaluation of air purification systems, an acrylic chamber was selected as the test environment [10, 11] . Testing was performed within a 240 L acrylic chamber that measures 100 cm (W) x 60 cm (D) x 40 cm (H). The acrylic chamber was placed within a Class II (Type A1) biological safety cabinet (BSC). As ozone is heavier than air, and therefore tends to sink, the inoculated gauze and steel plates were placed on a platform 30 cm higher than the floor of the acrylic chamber. In order to prevent the accumulation of ozone and ions within the acrylic chamber, the acrylic chamber was raised 2 cm above the BSC workspace to pull air out of the chamber. A Model 1200 ozone counter (Dairec, Inc., Kurashiki City, Japan) was placed within the acrylic chamber to monitor ozone levels within the negative pressure environment. The temperature within the test chamber was 21-22℃ and 38-50% relative ( Figure 1 ). The bacteria and fungi inoculated gauze pads, as well as the human coronavirus 229E inoculated stainless steel plates were placed within the vicinity of the AIO-2 bipolar ionizer and left at room temperature for up to 4 h. The distance between the gauze pads or stainless steel plates and the AIO-2 bipolar ionizer was 35 cm. As a control, inoculated gauze pads and stainless-steel plates were placed within another acrylic chamber without the AIO-2 ionizer. After 4 h, bacteria and fungi from each of the gauze pads were extracted by immersion in tubes containing 10 mL of sterile physiological saline and vortexed. Tenfold serial dilutions of the suspensions were performed using sterile physiological saline after which 0.1 mL of each of the dilutions was inoculated using a spreader onto either trypticase soy agar, anaerobic blood agar plate with 5% horse blood or potato dextrose agar depending on the organism. Following incubation, colony counts of 3 replicates were performed to determine the mean colony counts. As the infectivity of human coronavirus 229E decreases significantly after drying on various surfaces, exposure to bipolar ionisation was for 2 h [12] . The inoculated stainless-steel squares were then retrieved and immediately immersed in 2 mL of E-MEM without 10% FBS and vortexed followed by serial 10-fold dilution using E-MEM without 10% FBS. Quantitation of viable human coronavirus 229E was performed using 96-well tissue culture microtiter plates that were seeded with MRC-5 cells. After washing the tissue culture microtiter plates with E-MEM without 10% FBS, 100 µL of the E-MEM used to elute inoculated virus on stainless steel plates were inoculated onto MRC-5 cells. After allowing for absorption for 1 h at 37C under 5% CO2, E-MEM without 10% FBS, was removed from the tissue culture microtiter plates and 100 µL E-MEM containing 2% FBS, 100 U/mL of penicillin and 100 U/mL streptomycin were dispensed into each well and incubated for 7 days at 37 C under 5% CO2. Following incubation, the cell culture media was removed from the microtiter trays and MRC-5 cells were exposed to 70% ethanol for 20 minutes. Following fixation, MRC-5 cells were observed for cytopathic effect (CPE) to determine the TCID50 using the Behrens-Karber method. Log reduction between controls and post-exposure was calculated using the following equation: Log10(A/B, where A is the cfu/ml or TCID50/mL after treatment and B is cfu/mL or TCID50/mL before treatment [13] . To convert the log reduction to a J o u r n a l P r e -p r o o f percentage reduction, % reduction = (1 -10 -L ) x 100 was utilized, where L represents log reduction. As shown in Table 1 During the triplicate testing of the organisms, ozone ppm was less than 0.055 except for one experiment in which the ozone ppm was 0.066. The ozone concentration was monitored continuously during bi-polar ionization exposure and did not show fluctuation as the air within the acrylic chamber was continuously pulled out. Ozone also served as a surrogate for positive and negative ions to ensure that the concentration of ions within the chamber was constant. Since the beginning of the COVID-19 pandemic, a number of hospitals, schools, casinos, and sports arenas have implemented variants of bipolar ionisation as a technology to disinfect air and surfaces [14] . However, one manufacturer is currently the subject of a class action lawsuit which claims that the manufacturer falsely claimed that its technology was effective against SARS-CoV-2 [15] . The suit cites Boeing's technical assessment of bipolar ionisation, which concluded that the manufacturer's technology cannot clean the air at the level claimed by independent testing. Although, bipolar ionisation technology has been around for decades, the lack of many rigorous peer-reviewed studies makes it difficult to assess the effectiveness of this are in far excess of achievable levels in a real-world setting. In a study an electronic company uses to promote the effectiveness of its plasma cluster bipolar ionisation technology in reducing the concentration of aerosolized SARS-CoV-2, the study was conducted in a 3 L enclosed chamber [16] . The Occupational Safety and Health Administration (OSHA) has set official exposure limits for ozone at no more than 0.1 ppm average over an 8-h period, which is somewhat higher than the U.S. Environmental Protection Agency`s (EPA) position that ozone output of indoor medical devices to be no more than 0.05 ppm [17] . In this study, ozone levels were well below 0.1 ppm and, with the exception of one reading of 0.066 ppm, ozone levels were within EPA guidelines. Furthermore, exposure of the samples was minimized by elevating the testing platform 30 cm above the acrylic chamber floor as ozone tends to sink rather than rise. Effective ozone concentrations for microorganisms have been reported to be 0.23 to 2.29 ppm for bacteria, 3 to 5 ppm for molds, 0.02 to 0.26 for fungi and 0.2 to 4.1 ppm for viruses [18, 19, 20] . While the effect of ozone on the microorganisms tested in this study cannot be completely ruledout, the ozone levels measured were below levels reported to inactivate microorganisms. Our results showed a 94.4% to 99.9% log cfu/gauze decrease within 4 h for C. difficile, MDRP, MRSA and KPC-KP. As these bacteria are important pathogens associated with HAI and found in the healthcare environment, bipolar ionisation merits further examination as a technology to minimize transmission of infections. The relatively low inactivation of MDRA conflicts with a previous study that showed a significant decrease in acinetobacter infections as well as patient colonization in an intensive care unit during a 5.5-month period using a negative air ionisation technology [4] . A longer exposure to bipolar ionisation in our study may have demonstrated higher inactivation of MDRA. The testing environment in this study does not reflect a hospital ward or room; however, it would be difficult to conduct an experiment in the general environment which would introduce other variables such as contamination from environment microorganisms. The ultimate usefulness of this technology may need to be evaluated in a pre-and post-intervention study to compare HAI rates. It has been estimated that 1 in 25 hospitalised patients in the U.S. develops an infection associated with hospital care; furthermore, additional infections are seen in other healthcare settings [21] . According to the WHO, 7 and 10 patients, respectively, develop at least one HAI in developed and developing countries [22] . The relative contribution of fomites and droplets or aerosals in disease transmission in the healthcare J o u r n a l P r e -p r o o f 8 setting is not clear. Beyond currently established protocols such as personal protective equipment, aseptic technique, hand hygiene, environmental cleanliness, etc. to minimize HAI, bipolar ionisation systems to further reduce the risk of HAI merits assessment of effectiveness as HAI continue to occur despite the implementation of these infection control measures. Used in Large-scale ventilation systems worldwide, bipolar ionization could be a secret weapon in the war against COVID-19 Biological impact of small air ions The effect of surface charge, negative and bipolar ionization on the deposition of airborne bacteria Air ionisation and colonisation/infection with methicillin-resistant Staphylococcus aureus and Acinetobacter species in an intensive care unit Healthcare-associated infection impact with bioaerosol treatment and COVID-19 mitigation measures Cleaning indoor air using bi-polar ionization technology Stability of SARS-CoV-2 in different environmental conditions Evaluating the microbiocidal effectiveness of a microplasma discharge device against various bacteria including antimicrobialresistant bacteria and fungi A comparison of persistence of SARS-CoV-2 variants on stainless steel Computational models for reducing air pollution through the generation of negative ions Streamer Technology Survival of human coronaviruses 229E and OC43 in suspension and after drying on surfaces: a possible source of hospitalacquired infections Log and percent reductions in microbiology and antimicrobial testing Stark Country schools invest in air cleaning technology to prevent the spread of COVID-19 In the United States District Court for the District of Delaware. Plaintiff v. Global Plasma Solutions Plasmacluster Technology Demonstrates Effectiveness in Reducing Airborne Novel Coronavirus (SARS-CoV-2), a World First The National Institute for Occupational Safety and Health (NIOSH) Could ozone be an effective disinfection measure against the novel coronavirus (SARS-CoV-2)? Application of ozone for enhancing the microbiological safety and quality of foods: a review Bactericidal effects of high airborne ozone concentrations on Escherichia coli and Staphylococcus aureus World Health Organization. Healthcare-associated infections Authors' contribution IK preparation, review, and submission of the manuscript. IK, AK and HT contributed to data collection, data analysis. IK and HT contributed to data analysis and review and submission of the manuscript.