In Vitro Bactericidal and Virucidal Efficacy of Povidone-Iodine Gargle/Mouthwash Against Respiratory and Oral Tract Pathogens

In Vitro Bactericidal and Virucidal Efficacy of Povidone-Iodine Gargle/Mouthwash Against... Infect Dis Ther (2018) 7:249–259 https://doi.org/10.1007/s40121-018-0200-7 ORIGINAL RESEARCH In Vitro Bactericidal and Virucidal Efficacy of Povidone-Iodine Gargle/Mouthwash Against Respiratory and Oral Tract Pathogens . . . Maren Eggers Torsten Koburger-Janssen Markus Eickmann Juergen Zorn Received: February 13, 2018 / Published online: April 9, 2018 The Author(s) 2018 subtype H1N1 according to virucidal quantita- ABSTRACT tive suspension test EN14476. PVP-I 7% gar- gle/mouthwash was diluted 1:30 with water to a Introduction: Recent virus epidemics and ris- concentration of 0.23% (the recommended ing antibiotic resistance highlight the impor- concentration for ‘‘real-life’’ use in Japan) and tance of hygiene measures to prevent and tested at room temperature under clean condi- control outbreaks. We investigated the in vitro tions [0.3 g/l bovine serum albumin (BSA), bactericidal and virucidal efficacy of povidone- viruses only] and dirty conditions (3.0 g/l iodine (PVP-I) 7% gargle/mouthwash at defined BSA ? 3.0 ml/l erythrocytes) as an interfering dilution against oral and respiratory tract substance for defined contact times (minimum pathogens. 15 s). Rotavirus was tested without protein load. Methods: PVP-I was tested against Klebsiella A C 5 log (99.999%) decrease of bacteria pneumoniae and Streptococcus pneumoniae and C 4 log (99.99%) reduction in viral titre according to bactericidal quantitative suspen- represented effective bactericidal and virucidal sion test EN13727 and against severe acute res- activity, respectively, per European standards. piratory syndrome and Middle East respiratory Results: PVP-I gargle/mouthwash diluted 1:30 syndrome coronaviruses (SARS-CoV and MERS- (equivalent to a concentration of 0.23% PVP-I) CoV), rotavirus strain Wa and influenza virus A showed effective bactericidal activity against Enhanced digital features To view enhanced digital Klebsiella pneumoniae and Streptococcus pneumo- features for this article, go to https://doi.org/10.6084/ niae and rapidly inactivated SARS-CoV, MERS- m9.figshare.6027224. CoV, influenza virus A (H1N1) and rotavirus after 15 s of exposure. M. Eggers (&) Conclusion: PVP-I 7% gargle/mouthwash Labor Prof. Gisela Enders MVZ GbR, Stuttgart, showed rapid bactericidal activity and virucidal Germany e-mail: eggers@labor-enders.de efficacy in vitro at a concentration of 0.23% PVP-I and may provide a protective oropha- T. Koburger-Janssen ryngeal hygiene measure for individuals at high Hygiene Nord GmbH, Greifswald, Germany risk of exposure to oral and respiratory M. Eickmann pathogens. Institute for Virology, Philipps University of Funding: Mundipharma Research GmbH & Co. Marburg, Marburg, Germany KG (MRG). J. Zorn Mundipharma Research GmbH & Co.KG, Limburg, Germany 250 Infect Dis Ther (2018) 7:249–259 Keywords: Anti-infective agents; Local; through the air and inoculated into the eyes, Microbial sensitivity tests; Mouthwashes; nose and mouth at close range [12]. Consider- Povidone-iodine; Respiratory tract infections ing these modes of transmission, oral hygiene by gargling, together with hand washing and mask use [13], may be beneficial to help min- INTRODUCTION imise the risk of both community- and hospital- acquired respiratory infections. Gargling is also Antibiotic resistance is rising to dangerously deemed to bring about favourable effects high levels worldwide [1]. Oral and respiratory through removal of oral/pharyngeal protease tract infections caused by bacteria such as that helps viral replication [14]. Effectiveness of Streptococcus pneumoniae and Klebsiella pneumo- the antiseptic agent in killing pathogens is niae pose a particular threat because of the rise paramount in selecting gargles/mouthwashes of antibiotic-resistant strains, with vulnerable for protective hygiene and can be achieved by patient populations at high risk of infection ensuring that antiseptic agents pass a standard [2, 3]. Seasonal endemic viruses such as influ- bactericidal or virucidal activity test. A rapid enza are another significant cause of respiratory action is also desirable, as the length of time infection; worldwide, annual influenza epi- that individuals are willing or able to keep the demics are estimated to result in about 3–5 product in the oral cavity is limited. million cases of severe illness and about Povidone-iodine (PVP-I) is a broad-spectrum 250,000–500,000 deaths [4]. In addition to sea- antimicrobial that has been used in infection sonal endemic viruses, emerging and re-emerg- control and prevention for over 60 years [15] ing virus outbreaks such as severe acute and is available in various preparations for use respiratory syndrome and Middle East respira- as a disinfectant for the skin, hands and muco- tory syndrome coronaviruses (SARS-CoV and sal surfaces, as well as for wound treatment and MERS-CoV) require close contact for human-to- eye applications. PVP-I has well-established human transmission and can spread nosoco- general antimicrobial activity, demonstrating mially [5, 6]. Unlike the remaining four coron- in vitro efficacy against gram-positive, gram- aviruses, which are typically associated with negative and some spore-forming bacteria mild, self-limiting respiratory illness, SARS-CoV (clostridia, Bacillus spp.) and mycobacteria and MERS-CoV cause severe respiratory symp- [16–20] and a wide range of enveloped and non- toms and are associated with considerable enveloped viruses [21–23]. Recent in vitro mortality [7]. There is no vaccination or any studies have demonstrated rapid virucidal specific antiviral treatment available for SARS- activity of PVP-I products against Ebola virus, CoV and MERS-CoV. Outbreaks can, however, MERS-CoV and European reference enveloped be quickly and effectively controlled with pre- virus [modified vaccinia virus Ankara (MVA)] ventive strategies based upon early accurate [24, 25]. Considering the proven in vitro effi- viral diagnosis, knowledge of the current epi- cacy, gargling with PVP-I may be an effective demiological season and effective hygiene method of preventing the spread of respiratory practices to decrease the risk of transmission [8]. viruses when an individual is contaminated by Effective hand hygiene minimises transmis- the airborne/droplet route or after uptake via sion of pathogens from contaminated hands of the mouth (such as when touching the mouth an infected individual through either direct or food with contaminated hands). The benefit person-to-person contact or indirectly via con- of gargling with PVP-I has already been noted in tamination of surfaces [9, 10]. Respiratory Japanese clinical respiratory guidelines [26]. pathogens such as influenza are also transmit- This study investigated the in vitro bacteri- ted via airborne dispersion of small particle cidal and virucidal efficacy of PVP-I 7% gar- aerosols (B 5 lm) when an infected individual gle/mouthwash against relevant oral and breathes, coughs or sneezes [11], while respira- respiratory tract pathogens based on the Euro- tory syncytial viruses, SARS-CoV and MERS-CoV pean standards EN13727 [27] and EN14476 [28]. can be spread by large droplets propelled Infect Dis Ther (2018) 7:249–259 251 (BSA) ? 3.0 ml/l erythrocytes] as interfering sub- METHODS stance. After the specified contact time (15 and 30 s), a 1-ml aliquot was taken, and the bactericidal Antiseptic product performance against test activity in this portion was immediately neu- bacteria was performed according to bacterici- tralised with 3% Tween 80 ? 0.1% histidine ? dal quantitative suspension test 0.3% lecithin ? 0.5% sodium thiosulphate. For EN13727:2012 ? A2:2015 [27] and against each test suspension, two 1-ml samples were model viruses under defined test conditions, spread on at least two plates each. The number of including temperature, contact time and inter- surviving test organisms in the mixture was cal- fering substances, according to virucidal quan- culated for each sample and the reduction factor titative suspension test EN14476:2013/ (RF) determined with respect to the corresponding FprA1:2015 [28]. Testing was performed from 11 test suspension. A reduction of bacteria of C 5log February 2016 to 8 March 2016. This article (C 99.999%) compared with the control was con- does not contain any studies with human par- sidered to represent effective antibacterial efficacy ticipants or animals performed by any of the according to European standards. authors. Virucidal Testing Product Tested The test viruses were coronaviruses SARS (strain The antiseptic product tested was 7% PVP-I gar- Frankfurt) and MERS (HCoV-EMC/2012), influ- gle/mouthwash [brand-name Isodine, manufac- enza A virus (H1N1)pdm09 and non-enveloped tured by Fukuchi Pharmaceutical Co., Ltd., Japan human rotavirus strain Wa. The host cells used (bactericidal testing) and Mundipharma Phar- for the virus cultivation and suspension test maceuticals Ltd. (virucidal testing)]. The 7% PVP- were Vero E6 cells for SARS-CoV and MERS- I solution was diluted with water (2 ml ? 60 ml, CoV, Madin-Darby Canine Kidney (MDCK) cells equivalent to a concentration of 0.23% of the for influenza virus A subtype H1N1 and MA104 active ingredient) prior to testing, according to cells for human rotavirus strain Wa. the manufacturer’s instructions for use in Japan Inactivation tests were conducted once in [29]. When using cell cultures in antiseptic pro- accordance with EN14476:2013/FprA1:2015 duct testing, the target cells are often more sen- [28] at 20.0 ± 1.0 C. The virus suspension was sitive to the active ingredient. To overcome this, added to the product test solution under clean the test product was further tested at 1:10, 1:100 (0.3 g/l BSA) and dirty conditions (3.0 g/l and 1:1000 dilutions of the 7% solution in bac- BSA ? 3.0 ml/l erythrocytes) as interfering sub- tericidal testing (corresponding to concentra- stance, except for the rotavirus suspension, tions of 0.7%, 0.07% and 0.007% of PVP-I) and at which was tested without protein load (using 1:300 and 1:3000 dilutions in virucidal testing distilled water as the interfering substance). The (corresponding to concentrations of 0.023% and test assay comprised 100 ll virus suspension, 0.0023% of PVP-I). 100 ll interfering substance and 800 ll PVP-I product (at the defined dilutions). A virus con- Bactericidal Testing trol mixture was also assessed using distilled water in place of the test product. After the Both gram-positive (Streptococcus pneumoniae, specified contact time (15 s for SARS-CoV and DSM 24048, ATCC 49619) and gram-negative MERS-CoV, 15 and 30 s for influenza, and 15, (Klebsiella pneumoniae, DSM 16609) reference 30, 60 and 120 s for rotavirus), virucidal activity strains were tested. of the solution was immediately suppressed by Inactivation tests were conducted once in dilution with nine volumes of ice-cold medium accordance with EN13727:2012 ? A2:2015 [27]at (MEM ? 2.0% FCS) and serially diluted ten fold. 20.0 ± 1.0 C. Asuspensionoftestorganisms was Due to the immediate titration, no after-effect added to the product test solution under dirty of the test product could occur. For each test conditions [3.0 g/l bovine serum albumin 252 Infect Dis Ther (2018) 7:249–259 suspension, six wells (5 wells for SARS-CoV and RESULTS MERS-CoV) of a microtitre plate containing a confluent monolayer of host cells were inocu- Bactericidal Activity lated with 100 ll of test suspension, and the cells were incubated at 37.0 C in a humidified The log reduction factors produced by PVP-I atmosphere under 5.0% CO . 7% gargle/mouthwash at defined dilutions After incubation, the medium was removed. against Klebsiella pneumoniae and Streptococcus For staining of influenza virus or rotavirus infec- pneumoniae under dirty conditions are shown by tivity, cells were fixed for 10 min with ice-cold contact time in Table 1. Bacterial counts in the acetone/methanol (40:60) and then blocked with control samples were 7.61 log /ml for Klebsiella 1% BSA in phosphate-buffered saline (PBS) for pneumoniae and 7.34 log /ml for Streptococcus 30 min and stained by the immunoperoxidase pneumoniae. All bacterial counts were reduced method. For testing against influenza A, a mono- by between [ 5.20 and [ 5.47 log /ml (corre- clonal antibody (25 ll/well) (Chemicon, Temec- sponding to a reduction in bacterial count of ula, CA) against influenza A (MAB8251) was C 99.999%) after 15 s of contact time at PVP-I applied. After incubation for 30 min at 37 C, the concentrations of 0.7% (1:10 dilution) and plates were washed and incubated with secondary 0.23% (1:30, i.e., recommended dilution). The horseradish peroxidase-labelled anti-mouse anti- lower concentrations of 0.07% and 0.007% did body (anti-mouse-HRP, DakoCytomation, Ger- not reach the threshold reduction, except for many) and finally with 3-amino-9-ethylcarbazole the 0.07% solution against Streptococcus pneu- (AEC) substrate (Sigma, St. Louis, MO, USA). For moniae after 30 s. testing against rotavirus, a peroxidase-labeled polyclonal goat antibody (BT81-2998-04, Bio- Virucidal Activity trend, Koln, Germany) against human rotavirus was applied. After incubation for 30 min at 37 C, The log reduction factors produced by PVP-I the plates were washed and incubated with sec- 7% gargle/mouthwash at defined dilutions ondary horseradish peroxidase-labelled anti-goat against each test virus are shown by interfering antibody (Sigma-Aldrich, Germany) at a dilution substance and contact time in Table 2. The viral of 1:500 and finally with AEC substrate. For all titres present in the control samples under clean virus testing, AEC (dilution 1:500) was used to and dirty conditions, respectively, were 7.10 visualise antibody binding and infected cells were and 6.90 log TCID /ml for SARS-CoV, 6.90 10 50 stained red. Stained cells were examined with a and 7.10 log TCID /ml for MERS-CoV, 7.17 10 50 light microscope. The cells were examined and 7.50 log TCID /ml for influenza virus A 10 50 microscopically for cytopathic effects (CPE). SARS subtype H1N1 and 6.17 log TCID /ml for 10 50 or MERS infected cells were not stained, but rotavirus (under clean conditions). All viral examined microscopically for infectivity and titres were reduced by between 4.40 and cytopathic effects. 6.00 log TCID /ml (corresponding to a 10 50 The virus titres were determined using the reduction in viral titre of C 99.99% for all Spearman-Ka¨rber method [30, 31] and expres- viruses tested) after 15 s of contact time with sed as tissue culture infectious dose 50% PVP-I gargle at a concentration of 0.23% (1:30, (TCID /ml). The virucidal activity was deter- i.e., recommended dilution). The lower PVP-I mined by the difference of the logarithmic titre concentrations of 0.023% (1:300 dilution) and of the virus control minus the logarithmic titre 0.0023% (1:3000 dilution) that were tested of the test virus (D log TCID /ml). This dif- 10 50 against rotavirus and influenza did not reach a ference was given as an RF including its 95% log reduction in viral titre C 4, except for the confidence interval. A reduction in virus titre of 0.023% concentration against influenza under C 4log (corresponding to an inactivation of clean conditions. C 99.99%) was regarded as evidence of suffi- cient virucidal activity. The calculation was performed according to EN14476 [28]. Infect Dis Ther (2018) 7:249–259 253 Table 1 Bactericidal activity of povidone-iodine 7% oral solution against gram-positive and -negative bacteria under dirty conditions Bacteria Povidone-iodine concentration (%) Log reduction factor 15 s 30 s Klebsiella pneumoniae 0.7 > 5.47 > 5.47 0.23 5.35 > 5.47 0.07 \ 2.79 3.24 0.007 \ 2.79 \ 2.79 Streptococcus pneumoniae 0.7 > 5.20 > 5.20 0.23 > 5.20 > 5.20 0.07 4.86 > 5.20 0.007 \ 2.52 \ 2.52 Results shown in bold indicate bactericidal activity (C 5 log reduction factor compared with control) Dirty conditions: 3.0 g/l bovine serum albumin ? 3.0 ml/l erythrocytes similar rapid antimicrobial activity in previous DISCUSSION in vitro studies. Against bacteria, Shimizu et al. demonstrated complete efficacy of PVP-I 0.2% Oral and respiratory tract pathogens represent a solution against clinical isolates of Klebsiella significant threat to human health. Nosocomial pneumoniae, Serratia marcescens, Pseudomonas infections are widespread, especially among aeruginosa, Alcaligenes faecalis and Alcaligenes more vulnerable patients, and are important xylosoxydans within 30 s using a simple contributors to morbidity and mortality. A methodology (the broth turbidity method) [18]. growing number of bacterial infections are In another study, low concentrations of PVP-I becoming harder, and sometimes impossible, to gargle/mouthwash (0.23–0.47%) killed methi- treat as antibiotics become less effective, and cillin-resistant Staphylococcus aureus (MRSA) and vaccination against respiratory viruses either Pseudomonas aeruginosa, including multidrug- does not exist or has incomplete coverage. When resistant strains, within 15-60 s in the presence there is an emerging infectious disease outbreak, of oral organic matter from healthy volunteers, practicing appropriate hygiene is recommended while 0.02% benzethonium chloride (BEC) and for both healthcare workers and individuals to 0.002% chlorhexidine gluconate (CHG) were limit the spread of infection by breaking the ineffective [32]. In a study comparing the bac- transmission. Oral hygiene could further tericidal activities of three gargle/mouthwashes improve the success rate of hygiene measures, against isolate and standard strains of gram- especially against respiratory pathogens. positive (MRSA) and -negative bacteria (Pseu- The data from this in vitro study demon- domonas aeruginosa and Klebsiella pneumoniae), strated rapid bactericidal and virucidal activity PVP-I (diluted 15-, 30- and 60-fold) elicited of PVP-I gargle/mouthwash against all respira- rapid killing of all three strains after 30 s of tory pathogens tested according to European exposure, while cetylpyridinium chloride (CPC) standard requirements. The minimum 15-s was effective only against gram-negative strains contact time proved to be sufficient for PVP-I after 60 s of exposure, and CHG was ineffective 7% gargle/mouthwash to be effective at the [33]. recommended dilution in Japan of 1:30 In previous virucidal studies, PVP-I gargle (equivalent to a concentration of 0.23% of the was found to inactivate a panel of viruses active ingredient). PVP-I solution has shown 254 Infect Dis Ther (2018) 7:249–259 Table 2 Virucidal activity of povidone-iodine 7% oral solution against SARS-CoV, MERS-CoV, influenza virus A subtype H1N1 and rotavirus Virus Povidone-iodine Log reduction factor with 95% confidence interval concentration (%) a b Clean conditions Dirty conditions 15 s 30 s 60 s 120 s 15 s 30 s Influenza virus A subtype H1N1 0.23 5.67 – 0.43 5.67 – 0.42 n.d. n.d. 6.00 – 0.47 6.00 – 0.47 0.023 4.50 – 0.54 4.83 – 0.68 n.d. n.d. 0.33 ± 0.63 0.50 ± 0.65 0.0023 0.83 ± 0.54 1.00 ± 0.70 n.d. n.d. 0.17 ± 0.58 0.17 ± 0.58 SARS-CoV 0.23 4.60 – 0.80 n.d. n.d. n.d. 4.40 – 0.79 n.d. MERS-CoV 0.23 4.40 – 0.79 n.d. n.d. n.d. 4.40 – 0.87 n.d. Non-enveloped human rotavirus 0.23 ‡ 4.67 – 0.42 ‡ 4.67 – 0.42 ‡ 4.67 – 0.42 ‡ 4.67 – 0.42 n.d. n.d. strain Wa 0.023 1.83 ± 0.54 2.00 ± 0.60 2.00 ± 0.60 2.17 ± 0.61 n.d. n.d. 0.0023 - 0.33 ± 0.42 0.00 ± 0.60 0.17 ± 0.61 0.67 ± 0.42 n.d. n.d. Results shown in bold indicate virucidal activity (C 4 log reduction in viral titre compared with control) BSA bovine serum albumin, MERS-CoV Middle East respiratory syndrome coronavirus, n.d. not done, SARS-CoV severe acute respiratory syndrome coronavirus Clean conditions: 0.3 g/l BSA as interfering substance, except for rotavirus testing, which used distilled water Dirty conditions: 3.0 g/l BSA ? 3.0 ml/l erythrocytes as interfering substance Infect Dis Ther (2018) 7:249–259 255 including adenovirus, mumps, rotavirus, polio- rotavirus, which was tested without protein virus (types 1 and 3), coxsackie virus, rhi- load). Pathogens are eradicated by the active novirus, herpes simplex virus, rubella, measles, moiety (non PVP-bound ‘free’ iodine) being influenza and human immunodeficiency virus, released into solution from the PVP-I complex, while CHG, benzalkonium chloride (BAC), BEC penetrating the cell wall and inactivating cells and alkyldiaminoethyl-glycine hydrochloride by forming complexes with amino acids and (AEG) gargles were ineffective against aden- unsaturated fatty acids, resulting in impaired ovirus, poliovirus and rhinovirus [22]. Eggers protein synthesis and alteration of cell mem- et al. demonstrated the virucidal activity of branes [38]. This basic mechanism of action PVP-I 4% skin cleanser, 7.5% surgical scrub, leads to strong microbicidal activity expressed 10% solution and 3.2% PVP-I/alcohol solution by multiple modes of action that include the against Ebola virus and MVA and of PVP-I 7.5% disruption of microbial metabolic pathways, as surgical scrub, 4% skin cleanser and 1% gar- well as destabilisation of the structural compo- gle/mouthwash against MERS-CoV and MVA nents of cell membranes, causing irreversible within 15 s of application [24, 25]. Application damage to the pathogen [39]. of PVP-I products with concentrations of The results of this study suggest that the use of 0.23–1% for 1–2 min reduced SARS-CoV virus PVP-I gargle/mouthwash may be a useful pro- infectivity from 1.17 9 10 TCID /ml to below tective measure against oral and respiratory tract detectable levels in a study by Kariwa et al., infections. Indeed, following the H1N1 swine flu although shorter contact times were not inves- outbreak in 2009, Japan’s Ministry of Health, tigated [23]. Ito et al. reported a reduction in Labour and Welfare recommended daily gargling viral infectious titres of avian influenza A viru- as a protective hygiene measure to prevent upper ses (H5N1, H5N3, H7N7 and H9N2) to below respiratory tract infections (URTIs) [40], a prac- detectable limits by incubation for only 10 s tice supported by findings from studies that with six different PVP-I products including examined the role of gargling in both healthy 0.23% gargle and 0.23% throat spray [34]. The individuals and those with frequent or persistent anti-influenza activity of PVP-I involves inhibi- URTIs [14, 41, 42]. Limited clinical studies have tion of viral haemagglutinin binding activity been performed that used PVP-I gargle/mouth- and viral neuraminidase catalytic hydrolysis wash to reduce the incidence of respiratory [35]. In the present study, PVP-I oral solution at infections in different settings. Shiraishi and a concentration of 0.23% was also effective Nakagawa showed a mean reduction rate in against non-enveloped rotavirus without inter- bacterial count immediately after gargling of fering substance after 15 s of exposure, which is 99.4% for PVP-I in volunteers (compared with in contrast to in vitro work by Steinmann et al. 59.7% for CHG and 97.0% for CPC) and a sig- [36], in which 7.5% PVP-I handwash was not nificantly lower absence rate due to URTIs at a active against the non-enveloped viruses tested, Japanese middle school where the use of PVP-I and by Sauerbrei and Wutzler [37], in which gargle was encouraged compared with schools PVP-I took 5 min to inactivate polyomavirus where PVP-I gargle was not used [33]. In patients SV40 and adenovirus. with chronic respiratory diseases, gargling with In this study, the virucidal and bactericidal PVP-I was found to reduce the episodes of infec- activity of PVP-I gargle/mouthwash was evalu- tions with Pseudomonas aeruginosa, Staphylococ- ated within a short exposure time (15 s) to cus aureus (including MRSA) and Haemophilus reflect a similar or shorter time than the actual influenzae by half [41]. Studies of prophylactic use gargling time in real-life conditions, since the of PVP-I gargle in patients requiring intubation length of time that individuals are willing to have also shown significant reductions in keep an antiseptic product in the oral cavity is oropharyngeal bacterial counts [43]. Oral limited. PVP-I oral solution at a concentration hygiene using PVP-I may be of particular benefit of 0.23% was effective against all pathogens in certain patient groups such as immunocom- tested in this study after the minimum contact promised patients at risk of prolonged virus time of 15 s, regardless of protein load (except shedding (which can increase the potential for 256 Infect Dis Ther (2018) 7:249–259 resistance to antiviral drugs and for nosocomial tract infections observed in other in vitro and transmission), patients with influenza to reduce in vivo studies and (3) the established safety the risk of secondary bacterial infection (that profile of PVP-I from over 60 years of use, pro- may appear, e.g., as otitis media in children and vide a strong rationale for the use of PVP-I oral thus avoid the need for antibiotics) and possibly solution for protective oropharyngeal hygiene in hospitalised patients to prevent the spread of management for individuals at high risk of influenza during high season. exposure to oral and respiratory pathogens. The safety profile of PVP-I is well established. In contrast to other antiseptic agents, PVP-I oral care products do not lead to any irritation or ACKNOWLEDGEMENTS damage of the oral mucosa, even with prolonged use [44, 45]. Although measurable systemic iodine absorption may occur with the long-term Funding. Sponsorship for both studies and use of PVP-I, its clinical manifestation as thyroid article processing charges was funded by Mun- dysfunction is not very common [39]. dipharma Research GmbH & Co. KG (MRG). All A limitation of this work is that the clinical authors had full access to all of the data in this relevance of such in vitro test results remains study and take complete responsibility for the unclear and needs to be supported by further integrity of the data and accuracy of the data investigations to evaluate the impact of gargling analysis. with PVP-I in real-life and clinical settings, although for ethical reasons, clinical studies Editorial Assistance. Editorial assistance in involving highly infective and dangerous the preparation of this article was provided by pathogens may not be feasible. Furthermore, Karen Mower of Scientific Editorial and funded our testing was limited to a few key respiratory by MRG. microorganisms. We selected Streptococcus Authorship. All named authors meet the pneumoniae as the main cause of community- acquired pneumonia and meningitis and Kleb- International Committee of Medical Journal siella pneumoniae because, although not a com- Editors (ICMJE) criteria for authorship for this mon cause of respiratory tract infections, it is an article, take responsibility for the integrity of emerging cause of multidrug-resistant nosoco- the work as a whole and have given their mial infection. In addition, these species repre- approval for this version to be published. sent both gram-positive and -negative bacteria. Prior Presentation. This work was presented Although our study did not include other at the 32nd Annual Meeting of the Japanese common bacterial pathogens causing pneumo- Society for Infection Prevention and Control, nia such as Staphylococcus aureus, Pseudomonas Japan, 24–25 February 2017. aeruginosa, Haemophilus influenzae and Acineto- bacter baumannii, the efficacy of PVP-I against Disclosures. Hygiene Nord GmbH and Lab- these pathogens has already been demonstrated oratory Enders performed the studies on behalf in previous studies [16, 18, 32, 33, 41, 46]. of the sponsor, MRG. Maren Eggers was paid travel expenses and an honorarium by Mundi- CONCLUSION pharma Pte Ltd. Torsten Koburger-Janssen is an employee of Hygiene Nord GmbH. Markus In conclusion, our study results, taken together Eickmann has nothing to disclose. Juergen Zorn with (1) recommendations for gargling with is an employee of MRG. antiseptic mouthwash for the control of oral Compliance with Ethics Guidelines. This and respiratory tract infections, (2) the rapid article does not contain any studies with bactericidal and virucidal efficacy of povidone- human participants or animals performed by iodine, including PVP-I gargle/mouthwash, any of the authors. against pathogens causing oral and respiratory Infect Dis Ther (2018) 7:249–259 257 int/gpsc/5may/tools/9789241597906/en/.Accessed Open Access. This article is distributed Mar 2018. under the terms of the Creative Commons Attribution-NonCommercial 4.0 International 10. Boyce JM, Pittet D, Healthcare Infection Control License (http://creativecommons.org/licenses/ Practices Advisory Committee, HICPAC/SHEA/ APIC/IDSA Hand Hygiene Task Force. Guideline for by-nc/4.0/), which permits any noncommer- hand hygiene in health-care settings. 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Reduction of potential respiratory pathogens by oral hygienic treatment in patients undergoing endotracheal anesthesia. J Anesth. 2003;17:84–91. 32. Yoneyama A, Shimizu M, Tabata M, Yashiro J, Takata T, Hikida M. In vitro short-time killing activity of povidone-iodine (Isodine Gargle) in the 44. Madan PD, Sequeira PS, Shenoy K, Shetty J. The presence of oral organic matter. Dermatology. effect of three mouthwashes on radiation-induced 2006;212(Suppl 1):103–8. oral mucositis in patients with head and neck Infect Dis Ther (2018) 7:249–259 259 malignancies: a randomized control trial. J Cancer 46. Lanjri S, Uwingabiye J, Frikh M, et al. In vitro Res Ther. 2008;4:3–8. evaluation of the susceptibility of Acinetobacter baumannii isolates to antiseptics and disinfectants: 45. Rahn R, Adamietz IA, Boettcher HD, Schaefer V, comparison between clinical and environmental Reimer K, Fleischer W. Povidone-iodine to prevent isolates. Antimicrob Resist Infect Control. mucositis in patients during antineoplastic 2017;6:36. radiochemotherapy. Dermatology. 1997;195(Suppl 2):57–61. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Diseases and Therapy Springer Journals

In Vitro Bactericidal and Virucidal Efficacy of Povidone-Iodine Gargle/Mouthwash Against Respiratory and Oral Tract Pathogens

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Abstract

Infect Dis Ther (2018) 7:249–259 https://doi.org/10.1007/s40121-018-0200-7 ORIGINAL RESEARCH In Vitro Bactericidal and Virucidal Efficacy of Povidone-Iodine Gargle/Mouthwash Against Respiratory and Oral Tract Pathogens . . . Maren Eggers Torsten Koburger-Janssen Markus Eickmann Juergen Zorn Received: February 13, 2018 / Published online: April 9, 2018 The Author(s) 2018 subtype H1N1 according to virucidal quantita- ABSTRACT tive suspension test EN14476. PVP-I 7% gar- gle/mouthwash was diluted 1:30 with water to a Introduction: Recent virus epidemics and ris- concentration of 0.23% (the recommended ing antibiotic resistance highlight the impor- concentration for ‘‘real-life’’ use in Japan) and tance of hygiene measures to prevent and tested at room temperature under clean condi- control outbreaks. We investigated the in vitro tions [0.3 g/l bovine serum albumin (BSA), bactericidal and virucidal efficacy of povidone- viruses only] and dirty conditions (3.0 g/l iodine (PVP-I) 7% gargle/mouthwash at defined BSA ? 3.0 ml/l erythrocytes) as an interfering dilution against oral and respiratory tract substance for defined contact times (minimum pathogens. 15 s). Rotavirus was tested without protein load. Methods: PVP-I was tested against Klebsiella A C 5 log (99.999%) decrease of bacteria pneumoniae and Streptococcus pneumoniae and C 4 log (99.99%) reduction in viral titre according to bactericidal quantitative suspen- represented effective bactericidal and virucidal sion test EN13727 and against severe acute res- activity, respectively, per European standards. piratory syndrome and Middle East respiratory Results: PVP-I gargle/mouthwash diluted 1:30 syndrome coronaviruses (SARS-CoV and MERS- (equivalent to a concentration of 0.23% PVP-I) CoV), rotavirus strain Wa and influenza virus A showed effective bactericidal activity against Enhanced digital features To view enhanced digital Klebsiella pneumoniae and Streptococcus pneumo- features for this article, go to https://doi.org/10.6084/ niae and rapidly inactivated SARS-CoV, MERS- m9.figshare.6027224. CoV, influenza virus A (H1N1) and rotavirus after 15 s of exposure. M. Eggers (&) Conclusion: PVP-I 7% gargle/mouthwash Labor Prof. Gisela Enders MVZ GbR, Stuttgart, showed rapid bactericidal activity and virucidal Germany e-mail: eggers@labor-enders.de efficacy in vitro at a concentration of 0.23% PVP-I and may provide a protective oropha- T. Koburger-Janssen ryngeal hygiene measure for individuals at high Hygiene Nord GmbH, Greifswald, Germany risk of exposure to oral and respiratory M. Eickmann pathogens. Institute for Virology, Philipps University of Funding: Mundipharma Research GmbH & Co. Marburg, Marburg, Germany KG (MRG). J. Zorn Mundipharma Research GmbH & Co.KG, Limburg, Germany 250 Infect Dis Ther (2018) 7:249–259 Keywords: Anti-infective agents; Local; through the air and inoculated into the eyes, Microbial sensitivity tests; Mouthwashes; nose and mouth at close range [12]. Consider- Povidone-iodine; Respiratory tract infections ing these modes of transmission, oral hygiene by gargling, together with hand washing and mask use [13], may be beneficial to help min- INTRODUCTION imise the risk of both community- and hospital- acquired respiratory infections. Gargling is also Antibiotic resistance is rising to dangerously deemed to bring about favourable effects high levels worldwide [1]. Oral and respiratory through removal of oral/pharyngeal protease tract infections caused by bacteria such as that helps viral replication [14]. Effectiveness of Streptococcus pneumoniae and Klebsiella pneumo- the antiseptic agent in killing pathogens is niae pose a particular threat because of the rise paramount in selecting gargles/mouthwashes of antibiotic-resistant strains, with vulnerable for protective hygiene and can be achieved by patient populations at high risk of infection ensuring that antiseptic agents pass a standard [2, 3]. Seasonal endemic viruses such as influ- bactericidal or virucidal activity test. A rapid enza are another significant cause of respiratory action is also desirable, as the length of time infection; worldwide, annual influenza epi- that individuals are willing or able to keep the demics are estimated to result in about 3–5 product in the oral cavity is limited. million cases of severe illness and about Povidone-iodine (PVP-I) is a broad-spectrum 250,000–500,000 deaths [4]. In addition to sea- antimicrobial that has been used in infection sonal endemic viruses, emerging and re-emerg- control and prevention for over 60 years [15] ing virus outbreaks such as severe acute and is available in various preparations for use respiratory syndrome and Middle East respira- as a disinfectant for the skin, hands and muco- tory syndrome coronaviruses (SARS-CoV and sal surfaces, as well as for wound treatment and MERS-CoV) require close contact for human-to- eye applications. PVP-I has well-established human transmission and can spread nosoco- general antimicrobial activity, demonstrating mially [5, 6]. Unlike the remaining four coron- in vitro efficacy against gram-positive, gram- aviruses, which are typically associated with negative and some spore-forming bacteria mild, self-limiting respiratory illness, SARS-CoV (clostridia, Bacillus spp.) and mycobacteria and MERS-CoV cause severe respiratory symp- [16–20] and a wide range of enveloped and non- toms and are associated with considerable enveloped viruses [21–23]. Recent in vitro mortality [7]. There is no vaccination or any studies have demonstrated rapid virucidal specific antiviral treatment available for SARS- activity of PVP-I products against Ebola virus, CoV and MERS-CoV. Outbreaks can, however, MERS-CoV and European reference enveloped be quickly and effectively controlled with pre- virus [modified vaccinia virus Ankara (MVA)] ventive strategies based upon early accurate [24, 25]. Considering the proven in vitro effi- viral diagnosis, knowledge of the current epi- cacy, gargling with PVP-I may be an effective demiological season and effective hygiene method of preventing the spread of respiratory practices to decrease the risk of transmission [8]. viruses when an individual is contaminated by Effective hand hygiene minimises transmis- the airborne/droplet route or after uptake via sion of pathogens from contaminated hands of the mouth (such as when touching the mouth an infected individual through either direct or food with contaminated hands). The benefit person-to-person contact or indirectly via con- of gargling with PVP-I has already been noted in tamination of surfaces [9, 10]. Respiratory Japanese clinical respiratory guidelines [26]. pathogens such as influenza are also transmit- This study investigated the in vitro bacteri- ted via airborne dispersion of small particle cidal and virucidal efficacy of PVP-I 7% gar- aerosols (B 5 lm) when an infected individual gle/mouthwash against relevant oral and breathes, coughs or sneezes [11], while respira- respiratory tract pathogens based on the Euro- tory syncytial viruses, SARS-CoV and MERS-CoV pean standards EN13727 [27] and EN14476 [28]. can be spread by large droplets propelled Infect Dis Ther (2018) 7:249–259 251 (BSA) ? 3.0 ml/l erythrocytes] as interfering sub- METHODS stance. After the specified contact time (15 and 30 s), a 1-ml aliquot was taken, and the bactericidal Antiseptic product performance against test activity in this portion was immediately neu- bacteria was performed according to bacterici- tralised with 3% Tween 80 ? 0.1% histidine ? dal quantitative suspension test 0.3% lecithin ? 0.5% sodium thiosulphate. For EN13727:2012 ? A2:2015 [27] and against each test suspension, two 1-ml samples were model viruses under defined test conditions, spread on at least two plates each. The number of including temperature, contact time and inter- surviving test organisms in the mixture was cal- fering substances, according to virucidal quan- culated for each sample and the reduction factor titative suspension test EN14476:2013/ (RF) determined with respect to the corresponding FprA1:2015 [28]. Testing was performed from 11 test suspension. A reduction of bacteria of C 5log February 2016 to 8 March 2016. This article (C 99.999%) compared with the control was con- does not contain any studies with human par- sidered to represent effective antibacterial efficacy ticipants or animals performed by any of the according to European standards. authors. Virucidal Testing Product Tested The test viruses were coronaviruses SARS (strain The antiseptic product tested was 7% PVP-I gar- Frankfurt) and MERS (HCoV-EMC/2012), influ- gle/mouthwash [brand-name Isodine, manufac- enza A virus (H1N1)pdm09 and non-enveloped tured by Fukuchi Pharmaceutical Co., Ltd., Japan human rotavirus strain Wa. The host cells used (bactericidal testing) and Mundipharma Phar- for the virus cultivation and suspension test maceuticals Ltd. (virucidal testing)]. The 7% PVP- were Vero E6 cells for SARS-CoV and MERS- I solution was diluted with water (2 ml ? 60 ml, CoV, Madin-Darby Canine Kidney (MDCK) cells equivalent to a concentration of 0.23% of the for influenza virus A subtype H1N1 and MA104 active ingredient) prior to testing, according to cells for human rotavirus strain Wa. the manufacturer’s instructions for use in Japan Inactivation tests were conducted once in [29]. When using cell cultures in antiseptic pro- accordance with EN14476:2013/FprA1:2015 duct testing, the target cells are often more sen- [28] at 20.0 ± 1.0 C. The virus suspension was sitive to the active ingredient. To overcome this, added to the product test solution under clean the test product was further tested at 1:10, 1:100 (0.3 g/l BSA) and dirty conditions (3.0 g/l and 1:1000 dilutions of the 7% solution in bac- BSA ? 3.0 ml/l erythrocytes) as interfering sub- tericidal testing (corresponding to concentra- stance, except for the rotavirus suspension, tions of 0.7%, 0.07% and 0.007% of PVP-I) and at which was tested without protein load (using 1:300 and 1:3000 dilutions in virucidal testing distilled water as the interfering substance). The (corresponding to concentrations of 0.023% and test assay comprised 100 ll virus suspension, 0.0023% of PVP-I). 100 ll interfering substance and 800 ll PVP-I product (at the defined dilutions). A virus con- Bactericidal Testing trol mixture was also assessed using distilled water in place of the test product. After the Both gram-positive (Streptococcus pneumoniae, specified contact time (15 s for SARS-CoV and DSM 24048, ATCC 49619) and gram-negative MERS-CoV, 15 and 30 s for influenza, and 15, (Klebsiella pneumoniae, DSM 16609) reference 30, 60 and 120 s for rotavirus), virucidal activity strains were tested. of the solution was immediately suppressed by Inactivation tests were conducted once in dilution with nine volumes of ice-cold medium accordance with EN13727:2012 ? A2:2015 [27]at (MEM ? 2.0% FCS) and serially diluted ten fold. 20.0 ± 1.0 C. Asuspensionoftestorganisms was Due to the immediate titration, no after-effect added to the product test solution under dirty of the test product could occur. For each test conditions [3.0 g/l bovine serum albumin 252 Infect Dis Ther (2018) 7:249–259 suspension, six wells (5 wells for SARS-CoV and RESULTS MERS-CoV) of a microtitre plate containing a confluent monolayer of host cells were inocu- Bactericidal Activity lated with 100 ll of test suspension, and the cells were incubated at 37.0 C in a humidified The log reduction factors produced by PVP-I atmosphere under 5.0% CO . 7% gargle/mouthwash at defined dilutions After incubation, the medium was removed. against Klebsiella pneumoniae and Streptococcus For staining of influenza virus or rotavirus infec- pneumoniae under dirty conditions are shown by tivity, cells were fixed for 10 min with ice-cold contact time in Table 1. Bacterial counts in the acetone/methanol (40:60) and then blocked with control samples were 7.61 log /ml for Klebsiella 1% BSA in phosphate-buffered saline (PBS) for pneumoniae and 7.34 log /ml for Streptococcus 30 min and stained by the immunoperoxidase pneumoniae. All bacterial counts were reduced method. For testing against influenza A, a mono- by between [ 5.20 and [ 5.47 log /ml (corre- clonal antibody (25 ll/well) (Chemicon, Temec- sponding to a reduction in bacterial count of ula, CA) against influenza A (MAB8251) was C 99.999%) after 15 s of contact time at PVP-I applied. After incubation for 30 min at 37 C, the concentrations of 0.7% (1:10 dilution) and plates were washed and incubated with secondary 0.23% (1:30, i.e., recommended dilution). The horseradish peroxidase-labelled anti-mouse anti- lower concentrations of 0.07% and 0.007% did body (anti-mouse-HRP, DakoCytomation, Ger- not reach the threshold reduction, except for many) and finally with 3-amino-9-ethylcarbazole the 0.07% solution against Streptococcus pneu- (AEC) substrate (Sigma, St. Louis, MO, USA). For moniae after 30 s. testing against rotavirus, a peroxidase-labeled polyclonal goat antibody (BT81-2998-04, Bio- Virucidal Activity trend, Koln, Germany) against human rotavirus was applied. After incubation for 30 min at 37 C, The log reduction factors produced by PVP-I the plates were washed and incubated with sec- 7% gargle/mouthwash at defined dilutions ondary horseradish peroxidase-labelled anti-goat against each test virus are shown by interfering antibody (Sigma-Aldrich, Germany) at a dilution substance and contact time in Table 2. The viral of 1:500 and finally with AEC substrate. For all titres present in the control samples under clean virus testing, AEC (dilution 1:500) was used to and dirty conditions, respectively, were 7.10 visualise antibody binding and infected cells were and 6.90 log TCID /ml for SARS-CoV, 6.90 10 50 stained red. Stained cells were examined with a and 7.10 log TCID /ml for MERS-CoV, 7.17 10 50 light microscope. The cells were examined and 7.50 log TCID /ml for influenza virus A 10 50 microscopically for cytopathic effects (CPE). SARS subtype H1N1 and 6.17 log TCID /ml for 10 50 or MERS infected cells were not stained, but rotavirus (under clean conditions). All viral examined microscopically for infectivity and titres were reduced by between 4.40 and cytopathic effects. 6.00 log TCID /ml (corresponding to a 10 50 The virus titres were determined using the reduction in viral titre of C 99.99% for all Spearman-Ka¨rber method [30, 31] and expres- viruses tested) after 15 s of contact time with sed as tissue culture infectious dose 50% PVP-I gargle at a concentration of 0.23% (1:30, (TCID /ml). The virucidal activity was deter- i.e., recommended dilution). The lower PVP-I mined by the difference of the logarithmic titre concentrations of 0.023% (1:300 dilution) and of the virus control minus the logarithmic titre 0.0023% (1:3000 dilution) that were tested of the test virus (D log TCID /ml). This dif- 10 50 against rotavirus and influenza did not reach a ference was given as an RF including its 95% log reduction in viral titre C 4, except for the confidence interval. A reduction in virus titre of 0.023% concentration against influenza under C 4log (corresponding to an inactivation of clean conditions. C 99.99%) was regarded as evidence of suffi- cient virucidal activity. The calculation was performed according to EN14476 [28]. Infect Dis Ther (2018) 7:249–259 253 Table 1 Bactericidal activity of povidone-iodine 7% oral solution against gram-positive and -negative bacteria under dirty conditions Bacteria Povidone-iodine concentration (%) Log reduction factor 15 s 30 s Klebsiella pneumoniae 0.7 > 5.47 > 5.47 0.23 5.35 > 5.47 0.07 \ 2.79 3.24 0.007 \ 2.79 \ 2.79 Streptococcus pneumoniae 0.7 > 5.20 > 5.20 0.23 > 5.20 > 5.20 0.07 4.86 > 5.20 0.007 \ 2.52 \ 2.52 Results shown in bold indicate bactericidal activity (C 5 log reduction factor compared with control) Dirty conditions: 3.0 g/l bovine serum albumin ? 3.0 ml/l erythrocytes similar rapid antimicrobial activity in previous DISCUSSION in vitro studies. Against bacteria, Shimizu et al. demonstrated complete efficacy of PVP-I 0.2% Oral and respiratory tract pathogens represent a solution against clinical isolates of Klebsiella significant threat to human health. Nosocomial pneumoniae, Serratia marcescens, Pseudomonas infections are widespread, especially among aeruginosa, Alcaligenes faecalis and Alcaligenes more vulnerable patients, and are important xylosoxydans within 30 s using a simple contributors to morbidity and mortality. A methodology (the broth turbidity method) [18]. growing number of bacterial infections are In another study, low concentrations of PVP-I becoming harder, and sometimes impossible, to gargle/mouthwash (0.23–0.47%) killed methi- treat as antibiotics become less effective, and cillin-resistant Staphylococcus aureus (MRSA) and vaccination against respiratory viruses either Pseudomonas aeruginosa, including multidrug- does not exist or has incomplete coverage. When resistant strains, within 15-60 s in the presence there is an emerging infectious disease outbreak, of oral organic matter from healthy volunteers, practicing appropriate hygiene is recommended while 0.02% benzethonium chloride (BEC) and for both healthcare workers and individuals to 0.002% chlorhexidine gluconate (CHG) were limit the spread of infection by breaking the ineffective [32]. In a study comparing the bac- transmission. Oral hygiene could further tericidal activities of three gargle/mouthwashes improve the success rate of hygiene measures, against isolate and standard strains of gram- especially against respiratory pathogens. positive (MRSA) and -negative bacteria (Pseu- The data from this in vitro study demon- domonas aeruginosa and Klebsiella pneumoniae), strated rapid bactericidal and virucidal activity PVP-I (diluted 15-, 30- and 60-fold) elicited of PVP-I gargle/mouthwash against all respira- rapid killing of all three strains after 30 s of tory pathogens tested according to European exposure, while cetylpyridinium chloride (CPC) standard requirements. The minimum 15-s was effective only against gram-negative strains contact time proved to be sufficient for PVP-I after 60 s of exposure, and CHG was ineffective 7% gargle/mouthwash to be effective at the [33]. recommended dilution in Japan of 1:30 In previous virucidal studies, PVP-I gargle (equivalent to a concentration of 0.23% of the was found to inactivate a panel of viruses active ingredient). PVP-I solution has shown 254 Infect Dis Ther (2018) 7:249–259 Table 2 Virucidal activity of povidone-iodine 7% oral solution against SARS-CoV, MERS-CoV, influenza virus A subtype H1N1 and rotavirus Virus Povidone-iodine Log reduction factor with 95% confidence interval concentration (%) a b Clean conditions Dirty conditions 15 s 30 s 60 s 120 s 15 s 30 s Influenza virus A subtype H1N1 0.23 5.67 – 0.43 5.67 – 0.42 n.d. n.d. 6.00 – 0.47 6.00 – 0.47 0.023 4.50 – 0.54 4.83 – 0.68 n.d. n.d. 0.33 ± 0.63 0.50 ± 0.65 0.0023 0.83 ± 0.54 1.00 ± 0.70 n.d. n.d. 0.17 ± 0.58 0.17 ± 0.58 SARS-CoV 0.23 4.60 – 0.80 n.d. n.d. n.d. 4.40 – 0.79 n.d. MERS-CoV 0.23 4.40 – 0.79 n.d. n.d. n.d. 4.40 – 0.87 n.d. Non-enveloped human rotavirus 0.23 ‡ 4.67 – 0.42 ‡ 4.67 – 0.42 ‡ 4.67 – 0.42 ‡ 4.67 – 0.42 n.d. n.d. strain Wa 0.023 1.83 ± 0.54 2.00 ± 0.60 2.00 ± 0.60 2.17 ± 0.61 n.d. n.d. 0.0023 - 0.33 ± 0.42 0.00 ± 0.60 0.17 ± 0.61 0.67 ± 0.42 n.d. n.d. Results shown in bold indicate virucidal activity (C 4 log reduction in viral titre compared with control) BSA bovine serum albumin, MERS-CoV Middle East respiratory syndrome coronavirus, n.d. not done, SARS-CoV severe acute respiratory syndrome coronavirus Clean conditions: 0.3 g/l BSA as interfering substance, except for rotavirus testing, which used distilled water Dirty conditions: 3.0 g/l BSA ? 3.0 ml/l erythrocytes as interfering substance Infect Dis Ther (2018) 7:249–259 255 including adenovirus, mumps, rotavirus, polio- rotavirus, which was tested without protein virus (types 1 and 3), coxsackie virus, rhi- load). Pathogens are eradicated by the active novirus, herpes simplex virus, rubella, measles, moiety (non PVP-bound ‘free’ iodine) being influenza and human immunodeficiency virus, released into solution from the PVP-I complex, while CHG, benzalkonium chloride (BAC), BEC penetrating the cell wall and inactivating cells and alkyldiaminoethyl-glycine hydrochloride by forming complexes with amino acids and (AEG) gargles were ineffective against aden- unsaturated fatty acids, resulting in impaired ovirus, poliovirus and rhinovirus [22]. Eggers protein synthesis and alteration of cell mem- et al. demonstrated the virucidal activity of branes [38]. This basic mechanism of action PVP-I 4% skin cleanser, 7.5% surgical scrub, leads to strong microbicidal activity expressed 10% solution and 3.2% PVP-I/alcohol solution by multiple modes of action that include the against Ebola virus and MVA and of PVP-I 7.5% disruption of microbial metabolic pathways, as surgical scrub, 4% skin cleanser and 1% gar- well as destabilisation of the structural compo- gle/mouthwash against MERS-CoV and MVA nents of cell membranes, causing irreversible within 15 s of application [24, 25]. Application damage to the pathogen [39]. of PVP-I products with concentrations of The results of this study suggest that the use of 0.23–1% for 1–2 min reduced SARS-CoV virus PVP-I gargle/mouthwash may be a useful pro- infectivity from 1.17 9 10 TCID /ml to below tective measure against oral and respiratory tract detectable levels in a study by Kariwa et al., infections. Indeed, following the H1N1 swine flu although shorter contact times were not inves- outbreak in 2009, Japan’s Ministry of Health, tigated [23]. Ito et al. reported a reduction in Labour and Welfare recommended daily gargling viral infectious titres of avian influenza A viru- as a protective hygiene measure to prevent upper ses (H5N1, H5N3, H7N7 and H9N2) to below respiratory tract infections (URTIs) [40], a prac- detectable limits by incubation for only 10 s tice supported by findings from studies that with six different PVP-I products including examined the role of gargling in both healthy 0.23% gargle and 0.23% throat spray [34]. The individuals and those with frequent or persistent anti-influenza activity of PVP-I involves inhibi- URTIs [14, 41, 42]. Limited clinical studies have tion of viral haemagglutinin binding activity been performed that used PVP-I gargle/mouth- and viral neuraminidase catalytic hydrolysis wash to reduce the incidence of respiratory [35]. In the present study, PVP-I oral solution at infections in different settings. Shiraishi and a concentration of 0.23% was also effective Nakagawa showed a mean reduction rate in against non-enveloped rotavirus without inter- bacterial count immediately after gargling of fering substance after 15 s of exposure, which is 99.4% for PVP-I in volunteers (compared with in contrast to in vitro work by Steinmann et al. 59.7% for CHG and 97.0% for CPC) and a sig- [36], in which 7.5% PVP-I handwash was not nificantly lower absence rate due to URTIs at a active against the non-enveloped viruses tested, Japanese middle school where the use of PVP-I and by Sauerbrei and Wutzler [37], in which gargle was encouraged compared with schools PVP-I took 5 min to inactivate polyomavirus where PVP-I gargle was not used [33]. In patients SV40 and adenovirus. with chronic respiratory diseases, gargling with In this study, the virucidal and bactericidal PVP-I was found to reduce the episodes of infec- activity of PVP-I gargle/mouthwash was evalu- tions with Pseudomonas aeruginosa, Staphylococ- ated within a short exposure time (15 s) to cus aureus (including MRSA) and Haemophilus reflect a similar or shorter time than the actual influenzae by half [41]. Studies of prophylactic use gargling time in real-life conditions, since the of PVP-I gargle in patients requiring intubation length of time that individuals are willing to have also shown significant reductions in keep an antiseptic product in the oral cavity is oropharyngeal bacterial counts [43]. Oral limited. PVP-I oral solution at a concentration hygiene using PVP-I may be of particular benefit of 0.23% was effective against all pathogens in certain patient groups such as immunocom- tested in this study after the minimum contact promised patients at risk of prolonged virus time of 15 s, regardless of protein load (except shedding (which can increase the potential for 256 Infect Dis Ther (2018) 7:249–259 resistance to antiviral drugs and for nosocomial tract infections observed in other in vitro and transmission), patients with influenza to reduce in vivo studies and (3) the established safety the risk of secondary bacterial infection (that profile of PVP-I from over 60 years of use, pro- may appear, e.g., as otitis media in children and vide a strong rationale for the use of PVP-I oral thus avoid the need for antibiotics) and possibly solution for protective oropharyngeal hygiene in hospitalised patients to prevent the spread of management for individuals at high risk of influenza during high season. exposure to oral and respiratory pathogens. The safety profile of PVP-I is well established. In contrast to other antiseptic agents, PVP-I oral care products do not lead to any irritation or ACKNOWLEDGEMENTS damage of the oral mucosa, even with prolonged use [44, 45]. Although measurable systemic iodine absorption may occur with the long-term Funding. Sponsorship for both studies and use of PVP-I, its clinical manifestation as thyroid article processing charges was funded by Mun- dysfunction is not very common [39]. dipharma Research GmbH & Co. KG (MRG). All A limitation of this work is that the clinical authors had full access to all of the data in this relevance of such in vitro test results remains study and take complete responsibility for the unclear and needs to be supported by further integrity of the data and accuracy of the data investigations to evaluate the impact of gargling analysis. with PVP-I in real-life and clinical settings, although for ethical reasons, clinical studies Editorial Assistance. Editorial assistance in involving highly infective and dangerous the preparation of this article was provided by pathogens may not be feasible. Furthermore, Karen Mower of Scientific Editorial and funded our testing was limited to a few key respiratory by MRG. microorganisms. We selected Streptococcus Authorship. All named authors meet the pneumoniae as the main cause of community- acquired pneumonia and meningitis and Kleb- International Committee of Medical Journal siella pneumoniae because, although not a com- Editors (ICMJE) criteria for authorship for this mon cause of respiratory tract infections, it is an article, take responsibility for the integrity of emerging cause of multidrug-resistant nosoco- the work as a whole and have given their mial infection. In addition, these species repre- approval for this version to be published. sent both gram-positive and -negative bacteria. Prior Presentation. This work was presented Although our study did not include other at the 32nd Annual Meeting of the Japanese common bacterial pathogens causing pneumo- Society for Infection Prevention and Control, nia such as Staphylococcus aureus, Pseudomonas Japan, 24–25 February 2017. aeruginosa, Haemophilus influenzae and Acineto- bacter baumannii, the efficacy of PVP-I against Disclosures. Hygiene Nord GmbH and Lab- these pathogens has already been demonstrated oratory Enders performed the studies on behalf in previous studies [16, 18, 32, 33, 41, 46]. of the sponsor, MRG. Maren Eggers was paid travel expenses and an honorarium by Mundi- CONCLUSION pharma Pte Ltd. Torsten Koburger-Janssen is an employee of Hygiene Nord GmbH. Markus In conclusion, our study results, taken together Eickmann has nothing to disclose. Juergen Zorn with (1) recommendations for gargling with is an employee of MRG. antiseptic mouthwash for the control of oral Compliance with Ethics Guidelines. This and respiratory tract infections, (2) the rapid article does not contain any studies with bactericidal and virucidal efficacy of povidone- human participants or animals performed by iodine, including PVP-I gargle/mouthwash, any of the authors. against pathogens causing oral and respiratory Infect Dis Ther (2018) 7:249–259 257 int/gpsc/5may/tools/9789241597906/en/.Accessed Open Access. This article is distributed Mar 2018. under the terms of the Creative Commons Attribution-NonCommercial 4.0 International 10. Boyce JM, Pittet D, Healthcare Infection Control License (http://creativecommons.org/licenses/ Practices Advisory Committee, HICPAC/SHEA/ APIC/IDSA Hand Hygiene Task Force. Guideline for by-nc/4.0/), which permits any noncommer- hand hygiene in health-care settings. 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Infectious Diseases and TherapySpringer Journals

Published: Apr 9, 2018

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