Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga

Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga Hae-Jin Sohn, A-Young Park, Jeong-Heon Lee, Kyu-Hwa Yun, Kyoung-Ju Song, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3969220/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2024 Read the published version in Parasitology Research → Version 1 posted 7 You are reading this latest preprint version Abstract The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO 2 ) gas is a potent disinfectant that is harmless to humans. In this study, we examined the amoebicidal effects of ClO 2 gas on N. fowleri and A. polyphaga . These amoebae were exposed to ClO 2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO 2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO 2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO 2 gas. ClO 2 gas has an amoebicidal effect on N. fowleri and A. polyphaga . Therefore, ClO 2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination. Naegleri fowleri Acanthamoeba polyphaga chlorine dioxide (ClO2) gas disinfectant amoebicidal effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The ubiquitous free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. Naegleria fowleri can cause a sudden and severe brain infection known as primary amoebic meningoencephalitis (PAM) in humans and animals (Ma et al., 1990; Marciano-Cabral and Cabral, 2007; Visvesvara, 2013). Some Acanthamoeba spp. are the causative agents of severe medical complications including fatal granulomatous encephalitis (GAE) and Acanthamoeba keratitis (AK) (Lorenzo-Morales et al., 2015; Marciano-Cabral et al., 2000). Humans are often infected with N. fowleri through water activities and pollution and develop PAM, leading to serious medical and social problems (Marciano-Cabral, 1988; Siddiqui et al., 2016; Taravaud et al., 2018). The infection primarily occurs when contaminated water is inhaled through the nose; the amoeba enters the nasal cavity and olfactory nerve, traverses the central nervous system (CNS), and kills its host within 5–9 days (Marciano-Cabral and Cabral, 2007; Martinez et al., 2010; Schuster and Visvesvara, 2004). As PAM is acute, and cerebrospinal fluid (CSF) is the main diagnostic specimen, early diagnosis is difficult. Anti-fungal agents (amphotericin B and/or miltefosine) have been used as treatment drugs; however, their effects are often unsatisfactory. Consequently, PAM has a high mortality rate of 95% (Kim et al., 2008; Visvesvara et al., 2007). AK is a rare but painful and severe corneal infection often observed in patients who wear contact lenses (Marciano-Cabral and Cabral, 2003). As the diagnosis of AK is difficult and delayed, a rapid and accurate diagnosis method is urgently required for AK. Chlorine dioxide (ClO 2 ) is a highly effective disinfectant. It is a yellowish-green gas at room temperature that is relatively easily dissolved in water; therefore, it can be used in various forms. This compound is a free radical, strongly oxidizing disinfectant with a stronger biocidal capacity than chlorine-based disinfectants and alcohols. ClO 2 has a sterilizing effect over a broad pH range of 2–10 and is useful as a food-washing solution and disinfectant owing to its relative stability and high solubility. It eliminates pigments, oxidizes iron and manganese, and neutralizes odors (Vogt et al., 2010). ClO 2 gas is well-known for its outstanding deodorizing, sterilizing, and viral amplification inhibitory properties (Kaly-Kullai et al., 2020; O Young, 2016). It is cost-effective, safe, and has eco-friendly qualities such as rapid light-induced breakdown (Wood et al., 2010). ClO 2 gas has been extensively used to disinfect various medical equipment; without direct application to the surface of an object, the growth of many bacterial species in the immediate vicinity can be prevented. A previous study on the microbiocidal mechanism of a ClO 2 solution reported that the primary mechanism in the treatment of Escherichia coli was the disruption of protein synthesis (Benarde et al., 1967). Bacillus subtilis is also inactivated by free chlorine and ClO 2 solutions owing to alterations in cell permeability (Young and Setlow, 2003). ClO 2 applications have recently expanded to include food disinfection and medical therapy. Moreover, ClO 2 in the gas form is an excellent disinfectant that can be used in daily life at low concentrations. However, owing to technical challenges in maintaining a consistent concentration of ClO 2 gas for evaporation, its practical application is limited. Although ClO 2 solution has antimicrobial effects, the reports documenting these effects are limited. Furthermore, our understanding of the amoebicidal mechanism of ClO 2 gas is still lacking, especially in free-living amoebae. Therefore, in this study, we investigated the amoebicidal effect of ClO 2 gas on N. fowleri and A. polyphaga , which was confirmed by observing amoebic viability by cell counting and mRNA expression of the N. fowleri and A. polyphaga actin genes nf-actin and ap-actin , respectively. Materials and Methods Preparation of N. fowleri and A. polyphaga The trophozoites of N. fowleri (Carter NF69 strain, ATCC NO. 30215) and A. polyphaga were axenically cultured at 37 °C in Nelson's medium containing 10% fetal bovine serum and at 30 °C in PYG medium, respectively (Visvesvara and Balamuth, 1975; Willaert, 1971). The amoebae were washed twice with phosphate-buffered saline (PBS) and centrifuged at 1500 rpm for 3 min. The cell pellets of N. fowleri and A. polyphaga were suspended in Nelson’s medium (2 × 10 5 cells/mL) and PYG medium (2 × 10 5 cells/mL), respectively, and then placed in 40φ culture dishes. Exposure of N. fowleri and A. polyphaga to ClO 2 gas ClO 2 gas from ‘PURISTIC’, a ready-to-use product from Purgofarm Co. Ltd. (Yongin City, Korea), was activated and placed in a small chamber (210 mm high × 297 mm wide × 250 mm deep), and the concentration of ClO 2 gas was measured using a gas detector (C-16, Analytical Technology, Collegeville, PA, USA). To begin the experiment, we pre-activated PURISTIC for 5 h, and then exposed N. fowleri and A. polyphaga to ClO 2 gas at 0.36 ppmv/h for 12, 24, 36, and 48 h. A group of N. fowleri and A. polyphaga were maintained without ClO 2 treatment as a control. Viability observation During the experimental period, amoebic viability, which is demonstrated through morphological changes after ClO 2 gas treatment, was observed using an inverted microscope (Olympus, Tokyo, Japan). The viable ClO 2 gas-treated amoeba samples were re-cultured with fresh Nelson’s or PYG media to observe their ability to recover into trophozoites. Amoebic viability and growth rates were estimated by cell counting with a hemocytometer. Ultrastructural characteristics of N. fowleri and A. polyphaga trophozoites exposed to ClO 2 gas Amoebic trophozoites exposed to ClO 2 gas for 24 h in 40φ culture dishes were harvested, fixed with fixation buffer (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2), post fixed with 1% osmium tetroxide at room temperature, dehydrated with increasing concentrations of ethanol, and embedded in epoxy resin. Subsequently, the samples were cut into thin sections and stained with uranyl acetate and lead citrate and observed using a Sigma 500 transmission electron microscope (TEM; Carl Zeiss, Oberkochen, Germany). RT-PCR for the mRNA expression of actin gene in N. fowleri and A. polyphaga To confirm amoebic viability, reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the nf-actin (actin gene of N. fowleri ) and ap-actin (actin gene of A. polyphaga ) products according to a previously described method (Sohn et al., 2019). Amoebae at each stage of incubation were used to generate cDNA (5 µg), and total RNA was prepared using a Superscript First Strand synthesis system (Invitrogen, Carlsbad, CA, USA) and RNeasy®Mini kit (QIAGEN, Germantown, MD, USA), respectively. PCR conditions were as follows: 95°C for 5 min, 30 cycles at 95°C for 1 min, 50°C for 30 s, 72°C for 30 s, and a final extension for 10 min at 72°C. The amplified products were separated by electrophoresis on an ethidium bromide-stained 1% agarose gel and detected under UV light. Results Morphological changes of N. fowler and A. polyphaga exposed to ClO 2 gas An inverted microscope analysis revealed that N. fowleri exposed to ClO 2 gas showed some immobility after 3 h and became wrinkled in shape after 12 h (Fig. 1 ). At 12 h, N. fowleri trophozoites were converted to a precystic form (round shape). Unlike the N. fowleri trophozoites of the control group (no ClO 2 gas treatment), those in the treatment group were round, ruptured, and died after 24 h of treatment (Fig. 1 ). No amoeba trophozoites were detected after 36 h. N. fowleri exposed to ClO 2 gas could not be recovered in fresh Nelson’s medium (Fig. 1 ). In A. polyphaga , trophozoites of A. polyphaga exposed to ClO 2 gas showed some morphological changes and wrinkled forms after 24 h (Fig. 2 ). At 36 h, A. polyphaga trophozoites were converted to a precystic or cystic form. The trophozoites were rounded, ruptured, and died after 36 h of treatment, compared to those in the control group (Fig. 2 ). After 48 h, no trophozoites were detected and no recovery was observed in fresh PYG medium (Fig. 2 ). Ultrastructural characteristics of N. fowleri and A. polyphaga exposed to ClO 2 gas When N. fowleri and A. polyphaga were exposed to ClO 2 gas for 36 h, TEM analysis of the cellular response revealed increased breakdown of the nuclear membrane layers. With increased exposure to ClO 2 gas, trophozoites developed abnormal shapes and became fragmented. Ultrastructural changes within the trophozoites exposed to ClO 2 gas were investigated using TEM (Figs. 3 and 4 ). A loss of extracellular material was observed, which progressed to a loss of cellular membrane integrity in N. fowleri exposed to ClO 2 gas for 36 h (Fig. 3 ). When A. polyphaga was exposed to ClO 2 gas for 36 h, the trophozoites showed the same morphological changes as N. fowleri (Fig. 4 ). Cellular membrane destruction, cytoplasmic damage, and lipid droplets were evident in both cases (Figs. 3 and 4 ). Growth rates of N. fowleri and A. polyphaga exposed to ClO 2 gas After treatment with ClO 2 gas, the growth rate of N. fowleri trophozoites decreased remarkably at 24 h, showing an approximately 80% reduction compared to that of the control group (Fig. 5 A). At 24 h, the number of trophozoites of N. fowleri significantly decreased, and cysts appeared to increase slightly; however, the number of cysts also decreased at 36 and 48 h (Fig. 5 A). In A. polyphaga , the growth rate of trophozoites decreased after 36 h (Fig. 5 B). Similar to the results for N. fowleri , the number of cysts increased at 24 h and then decreased significantly at 36 and 48 h (Fig. 5 B). These results show that ClO 2 gas had cytotoxic effects on N. fowleri and A. polyphaga in a time-dependent manner (Fig. 5 ). mRNA expression of actin gene in N. fowleri and A. polyphaga exposed to ClO 2 gas RT-PCR was performed to confirm the viability of N. fowleri and A. polyphaga exposed to ClO 2 . The mRNA expression levels of nf-actin and ap-actin mRNA decreased in a time-dependent manner (Fig. 6 ). In particular, no nf - actin mRNA expression was observed in N. fowleri exposed to ClO 2 gas for 48 h (Fig. 6 A). Discussion Patients with PAM are mostly infected through various amoeba-contaminated freshwater sources, such as bathing in ponds and hot springs, diving and skiing in rivers, and drinking tap water (Chen et al., 2019; Izumiyama et al., 2003; Maclean et al., 2004; Marciano-Cabral, 1988). The disinfection or decontamination of freshwater contaminated with N. fowleri is still in its nascent stage. Additionally, PAM is difficult to diagnose, and no existing treatment has achieved satisfactory results (Kim et al., 2008; Visvesvara et al., 2007). In addition, the incidence of AK among patients who wear contact lenses has been increasing worldwide (Lorenzo-Morales et al., 2015). Acanthamoeba spp. infiltrate the corneal stroma and epithelial cells, which is followed by the progressive infiltration of inflammatory cells. Therefore, the removal of pathogenic N. fowleri and A. polyphaga from fresh and polluted water is important. The US Environmental Protection Agency (US EPA) first approved the liquid form of ClO 2 for use as a disinfectant and sanitizer at various sites, including animal farms, bottling plants, and food processing, handling, and storage plants, in 1967, under the authority of the Federal Insecticide, Fungicide, and Rodenticide Act (Benarde et al., 1967). In 1988, the US EPA approved ClO 2 gas as a disinfectant for use on tools, clean rooms, environmental surfaces, manufacturing, and laboratory equipment and in 2006, they published a re-registration eligibility decision regarding ClO 2 as a pesticide (EPAGuidanceManual, 2006). ClO 2 is rapidly decomposed to the byproducts chlorite (ClO 2 -) and chlorate (ClO 3 -) upon contact with naturally occurring organic and inorganic substances. Evaluation of these byproducts are reported to have acceptable daily intake (ADI) values of 0.03 mg/kg body weight (bw) per day for chlorite and 0.01 mg/kg bw per day for chlorate (‎JEFCA, 2007; 2008). Clinical research has shown that ClO 2 is an effective disinfectant and cytotoxic to bacteria, fungi, and viruses (Kuo-Shan Yao, 2010; Sanekata et al., 2010; Wei et al., 2008). ClO 2 gas is several times more potent than sodium hypochlorite and has been shown to be effective in various situations. Sifaoui et al. (Sifaoui et al., 2021) reported the effects of a commercial disinfectant (CLORICAN) on Acanthamoeba spp. and N. fowleri viability. They reported that CLORICAN, a commercial liquid form of chlorine dioxide used to disinfect swimming pools, had a stronger effect on N. fowleri than Acanthamoeba spp. (Sifaoui et al., 2021). In this study, the growth rate of N. fowleri trophozoites decreased remarkably after 24 h of exposure to ClO 2 gas, showing an approximately 80% reduction compared to the control group. In addition, the growth rate of A. polyphaga trophozoites decreased after 24 h of exposure to ClO 2 . In both cases, recovery of amoebae treated with ClO 2 gas was not possible. The cell membrane is an important structure in both prokaryotes and eukaryotes, and disruption of the nuclear and cytoplasmic membrane layers and cytoplasmic organelle degradation were observed by TEM analysis in both N. fowleri and A. polyphaga exposed to ClO 2 gas. These findings reveal its efficacy as a disinfectant, similar to the effects observed in bacteria, fungi, and viruses (Kuo-Shan Yao, 2010; Sanekata et al., 2010; Wei et al., 2008). In our study, ClO 2 gas was shown to have high sensitivity on both N. fowleri and A. polyphaga . ClO 2 gas alters the amoeboid shape and induces cell shrinkage. Additionally, we investigated actin gene expression in N. fowleri and A. polyphaga exposed to ClO 2 gas. The actin gene is a major component of the cytoskeleton of pathogenic free-living amoebae such as N. fowleri and A. polyphaga (Sohn et al., 2019). Overexpression of the actin genes in N. fowleri or A. polyphaga trophozoites increases their amoebic motility and phagocytosis (Sohn et al., 2010). Therefore, we analyzed the expression levels of actin mRNA using RT-PCR to confirm the physiological changes in N. fowleri and A. polyphaga treated with ClO 2 gas. In both cases, the levels of actin mRNA in N. fowleri and A. polyphaga exposed to ClO 2 gas decreased in a time-dependent manner. Specifically, no actin mRNA expression was observed in N. fowleri exposed to ClO 2 gas for 48 h, indicating that ClO 2 gas completely eliminated N. fowleri trophozoites. These results suggest that ClO 2 gas exerts amoebicidal effects on N. fowleri and A. polyphaga by reducing their motility and phagocytic activity. However, further studies are required to elucidate this mechanism. Therefore, ClO 2 gas has been proposed as an efficient agent for the prevention and control of pathogenic free-living amoebae such as N. fowleri and A. polyphaga . Declarations Acknowledgements We would like to thank to Jong-Rak Kim, CEO of Purgofarm Co. for providing the materials. A uthor contribution Conceptualization, Hae-Jin Sohn, Kyoung-Ju Song and Ho-Joon Shin; Data curation, Hae-Jin Sohn, A-Young Park, Kyu-Hwa Yun and Jong-Hyun Kim; Formal analysis, Hae-Jin Sohn, Jeong-Heon Lee and Kyu-Hwa Yun; Investigation, Hae-Jin Sohn; Methodology, Hae-Jin Sohn and A-Young Park; Resources, Kyoung-Ju Song; Software, Kyoung-Ju Song; Supervision, Ho-Joon Shin; Validation, Hae-Jin Sohn; Visualization, Hae-Jin Sohn, A-Young Park and Jeong-Heon Lee; Writing – original draft, Hae-Jin Sohn; Writing – review & editing, Jong-Hyun Kim and Ho-Joon Shin. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1C1C2009518). Data availability All data generated or analyzed during this study are included in this published article. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2024 Read the published version in Parasitology Research → Version 1 posted Editorial decision: Revision requested 27 Mar, 2024 Reviews received at journal 12 Mar, 2024 Reviewers agreed at journal 05 Mar, 2024 Reviewers invited by journal 05 Mar, 2024 Editor assigned by journal 23 Feb, 2024 Submission checks completed at journal 23 Feb, 2024 First submitted to journal 19 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3969220","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274511028,"identity":"2a5f8b59-e410-4207-a038-d52c6aba1d29","order_by":0,"name":"Hae-Jin Sohn","email":"","orcid":"","institution":"Ajou University School of medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hae-Jin","middleName":"","lastName":"Sohn","suffix":""},{"id":274511029,"identity":"c3df5331-88f1-46cb-96d6-308e7d98834a","order_by":1,"name":"A-Young Park","email":"","orcid":"","institution":"Ajou University School of medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"A-Young","middleName":"","lastName":"Park","suffix":""},{"id":274511030,"identity":"1dbb1287-77a5-4df1-a457-0ad883738c5f","order_by":2,"name":"Jeong-Heon Lee","email":"","orcid":"","institution":"Ajou University School of medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeong-Heon","middleName":"","lastName":"Lee","suffix":""},{"id":274511031,"identity":"dd82e367-4ed9-4a07-a92f-f293265c37b5","order_by":3,"name":"Kyu-Hwa Yun","email":"","orcid":"","institution":"Graduate School of Ajou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyu-Hwa","middleName":"","lastName":"Yun","suffix":""},{"id":274511032,"identity":"8cc8384a-c757-401a-a2e1-6de4e03b519d","order_by":4,"name":"Kyoung-Ju Song","email":"","orcid":"","institution":"Chunsu Mountain Medicinal Herb Research Association","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyoung-Ju","middleName":"","lastName":"Song","suffix":""},{"id":274511033,"identity":"1cc43f1c-9075-49ce-b2e3-1c64a3af38f7","order_by":5,"name":"Jong-Hyun Kim","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jong-Hyun","middleName":"","lastName":"Kim","suffix":""},{"id":274511034,"identity":"298b7988-fdb1-4dac-9598-4a3f223acaed","order_by":6,"name":"Ho-Joon Shin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACA4YDbAwJDDYMjA0wASK1pJGkhYENSB1GFiAAzBmPP3vwcMd5eeb2HgOGHzUMxuYNBLRYNpwxN0g8c9uwseeMAWPPMQYzmQOEHHbgDJtEYtttxsYZOQYMvA0MNhKEHGZw4PgzoJZz9iAtjH+J03LADKjlQCJICzPQFjMitJwBaUlObuw5VnBY5piEMWEtN44/k/zZZme7sb1548M3NTaGMwhpYZA4AKENGxgYgEyCdgABfwOElidC7SgYBaNgFIxQAABXiEHspxcWAgAAAABJRU5ErkJggg==","orcid":"","institution":"Ajou University School of medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ho-Joon","middleName":"","lastName":"Shin","suffix":""}],"badges":[],"createdAt":"2024-02-19 07:06:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3969220/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3969220/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00436-024-08215-z","type":"published","date":"2024-04-01T22:28:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51770191,"identity":"e6d89176-5f05-486d-9681-ed8de2e3352a","added_by":"auto","created_at":"2024-02-28 19:23:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":382105,"visible":true,"origin":"","legend":"\u003cp\u003eMorphologic observation of \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 12, 24, 36, and 48 h; \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites treated with ClO\u003csub\u003e2\u003c/sub\u003e gas became wrinkled in shape and consequently died in a time-dependent manner (400× magnification). Scale bar = 10 μm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage211.png","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/4952f967936d4d9a7365d214.png"},{"id":51771194,"identity":"e45eeef5-ee7b-44e1-8d51-9fa68f9f37be","added_by":"auto","created_at":"2024-02-28 19:31:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177480,"visible":true,"origin":"","legend":"\u003cp\u003eMorphologic observation of \u003cem\u003eA. polyphaga\u003c/em\u003e trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 12, 24, 36, and 48 h; \u003cem\u003eA. polyphaga\u003c/em\u003e trophozoites treated with ClO\u003csub\u003e2\u003c/sub\u003e gas became wrinkled in shape and consequently died in a time-dependent manner (400×). Scale bar = 10 μm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/861a29b9fdbdc2d32a42a393.jpeg"},{"id":51771984,"identity":"0def2f8b-dba1-4f18-badb-2fffa3c41a3e","added_by":"auto","created_at":"2024-02-28 19:39:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":358848,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron micrographs of \u003cem\u003eN. fowleri \u003c/em\u003etrophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas. (\u003cstrong\u003eA\u003c/strong\u003e) Unexposed cells showed typical amoebic morphology; the nuclei (N) exhibited a homogeneous nucleoplasm with a prominent nucleolus. Pseudopods (P), vacuoles (V) and several mitochondria (M) were identified. (\u003cstrong\u003eB\u003c/strong\u003e) Trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 36 h exhibited membrane damage. (\u003cstrong\u003eC\u003c/strong\u003e) The cellular membrane of the amoeba shows a loss in membrane continuity. Lipid droplets (LD) are present in the cytoplasm. Scale bar = 1 μm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/d31c6924d178d83ec8ddb1d0.jpeg"},{"id":51770194,"identity":"53d8e8e0-8854-4c89-8ae1-178dc4b018ab","added_by":"auto","created_at":"2024-02-28 19:23:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":437024,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron micrographs of \u003cem\u003eA. polyphaga\u003c/em\u003e trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas. (\u003cstrong\u003eA\u003c/strong\u003e) Unexposed cells showed typical amoebic morphology; the nuclei (N) exhibited a homogeneous nucleoplasm with a prominent nucleolus. Vacuoles (V) and several mitochondria (M) were identified. (\u003cstrong\u003eB\u003c/strong\u003e) Trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 36 h exhibited membrane damage. (\u003cstrong\u003eC\u003c/strong\u003e) The cellular membrane of the amoeba shows a loss in membrane continuity. Lipid droplets (LD) are present in the cytoplasm. Scale bar = 1 μm.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/6bd94db976771e5f9bb1b404.jpeg"},{"id":51770197,"identity":"6db3bfa5-c643-46d3-b5fe-dbdd733d29fb","added_by":"auto","created_at":"2024-02-28 19:23:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":118255,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of \u003cem\u003eN. fowleri\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e) and \u003cem\u003eA. polyphaga\u003c/em\u003e (\u003cstrong\u003eB\u003c/strong\u003e) treated with ClO\u003csub\u003e2\u003c/sub\u003e gas. Untreated control (●); trophozoite (■) and cyst (▲) treated with ClO\u003csub\u003e2\u003c/sub\u003e gas.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/97714c7b5dfeafdb93259451.jpeg"},{"id":51770196,"identity":"ec6d11cb-d765-4c08-bc7d-81b7b8cf493c","added_by":"auto","created_at":"2024-02-28 19:23:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":68242,"visible":true,"origin":"","legend":"\u003cp\u003eActin gene expression in \u003cem\u003eN. fowleri\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e) and \u003cem\u003eA. polyphaga\u003c/em\u003e (\u003cstrong\u003eB\u003c/strong\u003e) treated with ClO\u003csub\u003e2\u003c/sub\u003e gas for 12, 24, 36, and 48 h. Untreated control (-); ClO\u003csub\u003e2\u003c/sub\u003e gas (+).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/f3c910b656c604eeca88f71f.jpeg"},{"id":55690184,"identity":"485bb19d-fa63-42a7-9556-011e6e0ef084","added_by":"auto","created_at":"2024-05-01 22:28:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2524632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3969220/v1/7d6ce7b4-5870-4f99-8a0b-23db25955023.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe ubiquitous free-living amoebae, \u003cem\u003eNaegleria fowleri\u003c/em\u003e and \u003cem\u003eAcanthamoeba polyphaga\u003c/em\u003e are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. \u003cem\u003eNaegleria fowleri\u003c/em\u003e can cause a sudden and severe brain infection known as primary amoebic meningoencephalitis (PAM) in humans and animals (Ma et al., 1990; Marciano-Cabral and Cabral, 2007; Visvesvara, 2013). Some \u003cem\u003eAcanthamoeba\u003c/em\u003e spp. are the causative agents of severe medical complications including fatal granulomatous encephalitis (GAE) and \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis (AK) (Lorenzo-Morales et al., 2015; Marciano-Cabral et al., 2000).\u003c/p\u003e \u003cp\u003eHumans are often infected with \u003cem\u003eN. fowleri\u003c/em\u003e through water activities and pollution and develop PAM, leading to serious medical and social problems (Marciano-Cabral, 1988; Siddiqui et al., 2016; Taravaud et al., 2018). The infection primarily occurs when contaminated water is inhaled through the nose; the amoeba enters the nasal cavity and olfactory nerve, traverses the central nervous system (CNS), and kills its host within 5\u0026ndash;9 days (Marciano-Cabral and Cabral, 2007; Martinez et al., 2010; Schuster and Visvesvara, 2004). As PAM is acute, and cerebrospinal fluid (CSF) is the main diagnostic specimen, early diagnosis is difficult. Anti-fungal agents (amphotericin B and/or miltefosine) have been used as treatment drugs; however, their effects are often unsatisfactory. Consequently, PAM has a high mortality rate of 95% (Kim et al., 2008; Visvesvara et al., 2007). AK is a rare but painful and severe corneal infection often observed in patients who wear contact lenses (Marciano-Cabral and Cabral, 2003). As the diagnosis of AK is difficult and delayed, a rapid and accurate diagnosis method is urgently required for AK.\u003c/p\u003e \u003cp\u003eChlorine dioxide (ClO\u003csub\u003e2\u003c/sub\u003e) is a highly effective disinfectant. It is a yellowish-green gas at room temperature that is relatively easily dissolved in water; therefore, it can be used in various forms. This compound is a free radical, strongly oxidizing disinfectant with a stronger biocidal capacity than chlorine-based disinfectants and alcohols. ClO\u003csub\u003e2\u003c/sub\u003e has a sterilizing effect over a broad pH range of 2\u0026ndash;10 and is useful as a food-washing solution and disinfectant owing to its relative stability and high solubility. It eliminates pigments, oxidizes iron and manganese, and neutralizes odors (Vogt et al., 2010). ClO\u003csub\u003e2\u003c/sub\u003e gas is well-known for its outstanding deodorizing, sterilizing, and viral amplification inhibitory properties (Kaly-Kullai et al., 2020; O Young, 2016). It is cost-effective, safe, and has eco-friendly qualities such as rapid light-induced breakdown (Wood et al., 2010). ClO\u003csub\u003e2\u003c/sub\u003e gas has been extensively used to disinfect various medical equipment; without direct application to the surface of an object, the growth of many bacterial species in the immediate vicinity can be prevented.\u003c/p\u003e \u003cp\u003eA previous study on the microbiocidal mechanism of a ClO\u003csub\u003e2\u003c/sub\u003e solution reported that the primary mechanism in the treatment of \u003cem\u003eEscherichia coli\u003c/em\u003e was the disruption of protein synthesis (Benarde et al., 1967). \u003cem\u003eBacillus subtilis\u003c/em\u003e is also inactivated by free chlorine and ClO\u003csub\u003e2\u003c/sub\u003e solutions owing to alterations in cell permeability (Young and Setlow, 2003). ClO\u003csub\u003e2\u003c/sub\u003e applications have recently expanded to include food disinfection and medical therapy. Moreover, ClO\u003csub\u003e2\u003c/sub\u003e in the gas form is an excellent disinfectant that can be used in daily life at low concentrations. However, owing to technical challenges in maintaining a consistent concentration of ClO\u003csub\u003e2\u003c/sub\u003e gas for evaporation, its practical application is limited. Although ClO\u003csub\u003e2\u003c/sub\u003e solution has antimicrobial effects, the reports documenting these effects are limited. Furthermore, our understanding of the amoebicidal mechanism of ClO\u003csub\u003e2\u003c/sub\u003e gas is still lacking, especially in free-living amoebae. Therefore, in this study, we investigated the amoebicidal effect of ClO\u003csub\u003e2\u003c/sub\u003e gas on \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e, which was confirmed by observing amoebic viability by cell counting and mRNA expression of the \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e actin genes \u003cem\u003enf-actin\u003c/em\u003e and \u003cem\u003eap-actin\u003c/em\u003e, respectively.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe trophozoites of \u003cem\u003eN. fowleri\u003c/em\u003e (Carter NF69 strain, ATCC NO. 30215) and \u003cem\u003eA. polyphaga\u003c/em\u003e were axenically cultured at 37 \u0026deg;C in Nelson's medium containing 10% fetal bovine serum and at 30 \u0026deg;C in PYG medium, respectively (Visvesvara and Balamuth, 1975; Willaert, 1971). The amoebae were washed twice with phosphate-buffered saline (PBS) and centrifuged at 1500 rpm for 3 min. The cell pellets of \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e were suspended in Nelson\u0026rsquo;s medium (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) and PYG medium (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL), respectively, and then placed in 40φ culture dishes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExposure of\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e \u003cb\u003eto ClO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eClO\u003csub\u003e2\u003c/sub\u003e gas from \u0026lsquo;PURISTIC\u0026rsquo;, a ready-to-use product from Purgofarm Co. Ltd. (Yongin City, Korea), was activated and placed in a small chamber (210 mm high \u0026times; 297 mm wide \u0026times; 250 mm deep), and the concentration of ClO\u003csub\u003e2\u003c/sub\u003e gas was measured using a gas detector (C-16, Analytical Technology, Collegeville, PA, USA). To begin the experiment, we pre-activated PURISTIC for 5 h, and then exposed \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e to ClO\u003csub\u003e2\u003c/sub\u003e gas at 0.36 ppmv/h for 12, 24, 36, and 48 h. A group of \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e were maintained without ClO\u003csub\u003e2\u003c/sub\u003e treatment as a control.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eViability observation\u003c/h2\u003e \u003cp\u003eDuring the experimental period, amoebic viability, which is demonstrated through morphological changes after ClO\u003csub\u003e2\u003c/sub\u003e gas treatment, was observed using an inverted microscope (Olympus, Tokyo, Japan). The viable ClO\u003csub\u003e2\u003c/sub\u003e gas-treated amoeba samples were re-cultured with fresh Nelson\u0026rsquo;s or PYG media to observe their ability to recover into trophozoites. Amoebic viability and growth rates were estimated by cell counting with a hemocytometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eUltrastructural characteristics of\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e \u003cb\u003etrophozoites exposed to ClO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmoebic trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 24 h in 40φ culture dishes were harvested, fixed with fixation buffer (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2), post fixed with 1% osmium tetroxide at room temperature, dehydrated with increasing concentrations of ethanol, and embedded in epoxy resin. Subsequently, the samples were cut into thin sections and stained with uranyl acetate and lead citrate and observed using a Sigma 500 transmission electron microscope (TEM; Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRT-PCR for the mRNA expression of\u003c/b\u003e \u003cb\u003eactin\u003c/b\u003e \u003cb\u003egene in\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo confirm amoebic viability, reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the \u003cem\u003enf-actin\u003c/em\u003e (actin gene of \u003cem\u003eN. fowleri\u003c/em\u003e) and \u003cem\u003eap-actin\u003c/em\u003e (actin gene of \u003cem\u003eA. polyphaga\u003c/em\u003e) products according to a previously described method (Sohn et al., 2019). Amoebae at each stage of incubation were used to generate cDNA (5 \u0026micro;g), and total RNA was prepared using a Superscript First Strand synthesis system (Invitrogen, Carlsbad, CA, USA) and RNeasy\u0026reg;Mini kit (QIAGEN, Germantown, MD, USA), respectively. PCR conditions were as follows: 95\u0026deg;C for 5 min, 30 cycles at 95\u0026deg;C for 1 min, 50\u0026deg;C for 30 s, 72\u0026deg;C for 30 s, and a final extension for 10 min at 72\u0026deg;C. The amplified products were separated by electrophoresis on an ethidium bromide-stained 1% agarose gel and detected under UV light.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMorphological changes of\u003c/b\u003e \u003cb\u003eN. fowler\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e \u003cb\u003eexposed to ClO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAn inverted microscope analysis revealed that \u003cem\u003eN. fowleri\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas showed some immobility after 3 h and became wrinkled in shape after 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At 12 h, \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites were converted to a precystic form (round shape). Unlike the \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites of the control group (no ClO\u003csub\u003e2\u003c/sub\u003e gas treatment), those in the treatment group were round, ruptured, and died after 24 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No amoeba trophozoites were detected after 36 h. \u003cem\u003eN. fowleri\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas could not be recovered in fresh Nelson\u0026rsquo;s medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In \u003cem\u003eA. polyphaga\u003c/em\u003e, trophozoites of \u003cem\u003eA. polyphaga\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas showed some morphological changes and wrinkled forms after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 36 h, \u003cem\u003eA. polyphaga\u003c/em\u003e trophozoites were converted to a precystic or cystic form. The trophozoites were rounded, ruptured, and died after 36 h of treatment, compared to those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After 48 h, no trophozoites were detected and no recovery was observed in fresh PYG medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eUltrastructural characteristics of\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e \u003cb\u003eexposed to ClO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhen \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e were exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 36 h, TEM analysis of the cellular response revealed increased breakdown of the nuclear membrane layers. With increased exposure to ClO\u003csub\u003e2\u003c/sub\u003e gas, trophozoites developed abnormal shapes and became fragmented. Ultrastructural changes within the trophozoites exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas were investigated using TEM (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A loss of extracellular material was observed, which progressed to a loss of cellular membrane integrity in \u003cem\u003eN. fowleri\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 36 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When \u003cem\u003eA. polyphaga\u003c/em\u003e was exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 36 h, the trophozoites showed the same morphological changes as \u003cem\u003eN. fowleri\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Cellular membrane destruction, cytoplasmic damage, and lipid droplets were evident in both cases (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth rates of\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e \u003cb\u003eexposed to ClO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter treatment with ClO\u003csub\u003e2\u003c/sub\u003e gas, the growth rate of \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites decreased remarkably at 24 h, showing an approximately 80% reduction compared to that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At 24 h, the number of trophozoites of \u003cem\u003eN. fowleri\u003c/em\u003e significantly decreased, and cysts appeared to increase slightly; however, the number of cysts also decreased at 36 and 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In \u003cem\u003eA. polyphaga\u003c/em\u003e, the growth rate of trophozoites decreased after 36 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similar to the results for \u003cem\u003eN. fowleri\u003c/em\u003e, the number of cysts increased at 24 h and then decreased significantly at 36 and 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results show that ClO\u003csub\u003e2\u003c/sub\u003e gas had cytotoxic effects on \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003emRNA expression of\u003c/b\u003e \u003cb\u003eactin\u003c/b\u003e \u003cb\u003egene in\u003c/b\u003e \u003cb\u003eN. fowleri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eA. polyphaga\u003c/b\u003e \u003cb\u003eexposed to ClO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003egas\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRT-PCR was performed to confirm the viability of \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e. The mRNA expression levels of \u003cem\u003enf-actin\u003c/em\u003e and \u003cem\u003eap-actin\u003c/em\u003e mRNA decreased in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In particular, no \u003cem\u003enf\u003c/em\u003e-\u003cem\u003eactin\u003c/em\u003e mRNA expression was observed in \u003cem\u003eN. fowleri\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003ePatients with PAM are mostly infected through various amoeba-contaminated freshwater sources, such as bathing in ponds and hot springs, diving and skiing in rivers, and drinking tap water (Chen et al., 2019; Izumiyama et al., 2003; Maclean et al., 2004; Marciano-Cabral, 1988). The disinfection or decontamination of freshwater contaminated with \u003cem\u003eN. fowleri\u003c/em\u003e is still in its nascent stage. Additionally, PAM is difficult to diagnose, and no existing treatment has achieved satisfactory results (Kim et al., 2008; Visvesvara et al., 2007). In addition, the incidence of AK among patients who wear contact lenses has been increasing worldwide (Lorenzo-Morales et al., 2015). \u003cem\u003eAcanthamoeba\u003c/em\u003e spp. infiltrate the corneal stroma and epithelial cells, which is followed by the progressive infiltration of inflammatory cells. Therefore, the removal of pathogenic \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e from fresh and polluted water is important.\u003c/p\u003e \u003cp\u003eThe US Environmental Protection Agency (US EPA) first approved the liquid form of ClO\u003csub\u003e2\u003c/sub\u003e for use as a disinfectant and sanitizer at various sites, including animal farms, bottling plants, and food processing, handling, and storage plants, in 1967, under the authority of the Federal Insecticide, Fungicide, and Rodenticide Act (Benarde et al., 1967). In 1988, the US EPA approved ClO\u003csub\u003e2\u003c/sub\u003e gas as a disinfectant for use on tools, clean rooms, environmental surfaces, manufacturing, and laboratory equipment and in 2006, they published a re-registration eligibility decision regarding ClO\u003csub\u003e2\u003c/sub\u003e as a pesticide (EPAGuidanceManual, 2006). ClO\u003csub\u003e2\u003c/sub\u003e is rapidly decomposed to the byproducts chlorite (ClO\u003csub\u003e2\u003c/sub\u003e-) and chlorate (ClO\u003csub\u003e3\u003c/sub\u003e-) upon contact with naturally occurring organic and inorganic substances. Evaluation of these byproducts are reported to have acceptable daily intake (ADI) values of 0.03 mg/kg body weight (bw) per day for chlorite and 0.01 mg/kg bw per day for chlorate (\u0026lrm;JEFCA, 2007; 2008). Clinical research has shown that ClO\u003csub\u003e2\u003c/sub\u003e is an effective disinfectant and cytotoxic to bacteria, fungi, and viruses (Kuo-Shan Yao, 2010; Sanekata et al., 2010; Wei et al., 2008). ClO\u003csub\u003e2\u003c/sub\u003e gas is several times more potent than sodium hypochlorite and has been shown to be effective in various situations.\u003c/p\u003e \u003cp\u003eSifaoui et al. (Sifaoui et al., 2021) reported the effects of a commercial disinfectant (CLORICAN) on \u003cem\u003eAcanthamoeba\u003c/em\u003e spp. and \u003cem\u003eN. fowleri\u003c/em\u003e viability. They reported that CLORICAN, a commercial liquid form of chlorine dioxide used to disinfect swimming pools, had a stronger effect on \u003cem\u003eN. fowleri\u003c/em\u003e than \u003cem\u003eAcanthamoeba\u003c/em\u003e spp. (Sifaoui et al., 2021). In this study, the growth rate of \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites decreased remarkably after 24 h of exposure to ClO\u003csub\u003e2\u003c/sub\u003e gas, showing an approximately 80% reduction compared to the control group. In addition, the growth rate of \u003cem\u003eA. polyphaga\u003c/em\u003e trophozoites decreased after 24 h of exposure to ClO\u003csub\u003e2\u003c/sub\u003e. In both cases, recovery of amoebae treated with ClO\u003csub\u003e2\u003c/sub\u003e gas was not possible. The cell membrane is an important structure in both prokaryotes and eukaryotes, and disruption of the nuclear and cytoplasmic membrane layers and cytoplasmic organelle degradation were observed by TEM analysis in both \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas. These findings reveal its efficacy as a disinfectant, similar to the effects observed in bacteria, fungi, and viruses (Kuo-Shan Yao, 2010; Sanekata et al., 2010; Wei et al., 2008). In our study, ClO\u003csub\u003e2\u003c/sub\u003e gas was shown to have high sensitivity on both \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e. ClO\u003csub\u003e2\u003c/sub\u003e gas alters the amoeboid shape and induces cell shrinkage.\u003c/p\u003e \u003cp\u003eAdditionally, we investigated \u003cem\u003eactin\u003c/em\u003e gene expression in \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas. The \u003cem\u003eactin\u003c/em\u003e gene is a major component of the cytoskeleton of pathogenic free-living amoebae such as \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e (Sohn et al., 2019). Overexpression of the \u003cem\u003eactin\u003c/em\u003e genes in \u003cem\u003eN. fowleri\u003c/em\u003e or \u003cem\u003eA. polyphaga\u003c/em\u003e trophozoites increases their amoebic motility and phagocytosis (Sohn et al., 2010). Therefore, we analyzed the expression levels of \u003cem\u003eactin\u003c/em\u003e mRNA using RT-PCR to confirm the physiological changes in \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e treated with ClO\u003csub\u003e2\u003c/sub\u003e gas. In both cases, the levels of \u003cem\u003eactin\u003c/em\u003e mRNA in \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas decreased in a time-dependent manner. Specifically, no \u003cem\u003eactin\u003c/em\u003e mRNA expression was observed in \u003cem\u003eN. fowleri\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 48 h, indicating that ClO\u003csub\u003e2\u003c/sub\u003e gas completely eliminated \u003cem\u003eN. fowleri\u003c/em\u003e trophozoites. These results suggest that ClO\u003csub\u003e2\u003c/sub\u003e gas exerts amoebicidal effects on \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e by reducing their motility and phagocytic activity. However, further studies are required to elucidate this mechanism. Therefore, ClO\u003csub\u003e2\u003c/sub\u003e gas has been proposed as an efficient agent for the prevention and control of pathogenic free-living amoebae such as \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank to Jong-Rak Kim, CEO of Purgofarm Co. for providing the materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econtribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Hae-Jin Sohn, Kyoung-Ju Song and Ho-Joon Shin; Data curation, Hae-Jin Sohn, A-Young Park, Kyu-Hwa Yun and Jong-Hyun Kim; Formal analysis, Hae-Jin Sohn, Jeong-Heon Lee and Kyu-Hwa Yun; Investigation, Hae-Jin Sohn; Methodology, Hae-Jin Sohn and A-Young Park; Resources, Kyoung-Ju Song; Software, Kyoung-Ju Song; Supervision, Ho-Joon Shin; Validation, Hae-Jin Sohn; Visualization, Hae-Jin Sohn, A-Young Park and Jeong-Heon Lee; Writing \u0026ndash; original draft, Hae-Jin Sohn; Writing \u0026ndash; review \u0026amp; editing, Jong-Hyun Kim and Ho-Joon Shin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1C1C2009518).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBenarde, M.A., Snow, W.B., Olivieri, V.P. and Davidson, B. 1967. Kinetics and mechanism of bacterial disinfection by chlorine dioxide. Appl Microbiol 15(2), 257\u0026ndash;265.\u003c/li\u003e\n \u003cli\u003eChen, M., Ruan, W., Zhang, L., Hu, B. and Yang, X. 2019. Primary Amebic Meningoencephalitis: A Case Report. Korean J Parasitol 57(3), 291\u0026ndash;294.\u003c/li\u003e\n \u003cli\u003eEPAGuidanceManual 2006 Chlorine dioxide and Sodium Chlorite. US Environmental Protection Agency.\u003c/li\u003e\n \u003cli\u003eIzumiyama, S., Yagita, K., Furushima-Shimogawara, R., Asakura, T., Karasudani, T. and Endo, T. 2003. Occurrence and distribution of Naegleria species in thermal waters in Japan. J Eukaryot Microbiol 50 Suppl, 514\u0026ndash;515.\u003c/li\u003e\n \u003cli\u003e\u0026lrm;JEFCA 2007 Evaluation of certain Food Contaminants. Sixty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives. Organization, W.H. (ed).\u003c/li\u003e\n \u003cli\u003e\u0026lrm;JEFCA 2008 Safety evaluation of certain food additives and contaminants / prepared by the sixty-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (\u0026lrm;JEFCA)\u0026lrm;. Nations, W.H.O.F.a.A.O.o.t.U. (ed).\u003c/li\u003e\n \u003cli\u003eKaly-Kullai, K., Wittmann, M., Noszticzius, Z. and Rosivall, L. 2020. Can chlorine dioxide prevent the spreading of coronavirus or other viral infections? Medical hypotheses. Physiol Int 107(1), 1\u0026ndash;11.\u003c/li\u003e\n \u003cli\u003eKim, J.H., Jung, S.Y., Lee, Y.J., Song, K.J., Kwon, D., Kim, K., Park, S., Im, K.I. and Shin, H.J. 2008. Effect of therapeutic chemical agents in vitro and on experimental meningoencephalitis due to Naegleria fowleri. Antimicrob Agents Chemother 52(11), 4010\u0026ndash;4016.\u003c/li\u003e\n \u003cli\u003eKuo-Shan Yao, Y.-H.H., Yu-Jie Chang, Chen-Yu Chang, Ta-Chih Cheng and Hui-Lin Liao 2010. Inactivation effect of chlorine dioxide on phytopathogenic bacteria in irrigation water. Journal of Environmental Engineering and Management.\u003c/li\u003e\n \u003cli\u003eLorenzo-Morales, J., Khan, N.A. and Walochnik, J. 2015. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite 22, 10.\u003c/li\u003e\n \u003cli\u003eMa, P., Visvesvara, G.S., Martinez, A.J., Theodore, F.H., Daggett, P.M. and Sawyer, T.K. 1990. Naegleria and Acanthamoeba infections: review. Rev Infect Dis 12(3), 490\u0026ndash;513.\u003c/li\u003e\n \u003cli\u003eMaclean, R.C., Richardson, D.J., LePardo, R. and Marciano-Cabral, F. 2004. The identification of Naegleria fowleri from water and soil samples by nested PCR. Parasitol Res 93(3), 211\u0026ndash;217.\u003c/li\u003e\n \u003cli\u003eMarciano-Cabral, F. 1988. Biology of Naegleria spp. Microbiol Rev 52(1), 114\u0026ndash;133.\u003c/li\u003e\n \u003cli\u003eMarciano-Cabral, F. and Cabral, G. 2003. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16(2), 273\u0026ndash;307.\u003c/li\u003e\n \u003cli\u003eMarciano-Cabral, F. and Cabral, G.A. 2007. The immune response to Naegleria fowleri amebae and pathogenesis of infection. FEMS Immunol Med Microbiol 51(2), 243\u0026ndash;259.\u003c/li\u003e\n \u003cli\u003eMarciano-Cabral, F., Puffenbarger, R. and Cabral, G.A. 2000. The increasing importance of Acanthamoeba infections. J Eukaryot Microbiol 47(1), 29\u0026ndash;36.\u003c/li\u003e\n \u003cli\u003eMartinez, D.Y., Seas, C., Bravo, F., Legua, P., Ramos, C., Cabello, A.M. and Gotuzzo, E. 2010. Successful treatment of Balamuthia mandrillaris amoebic infection with extensive neurological and cutaneous involvement. Clin Infect Dis 51(2), e7-11.\u003c/li\u003e\n \u003cli\u003eO Young, R. 2016. Chlorine Dioxide (CLO2) As a Non-Toxic Antimicrobial Agent for Virus, Bacteria and Yeast (Candida Albicans). International Journal of Vaccines \u0026amp; Vaccination 2(6).\u003c/li\u003e\n \u003cli\u003eSanekata, T., Fukuda, T., Miura, T., Morino, H., Lee, C., Maeda, K., Araki, K., Otake, T., Kawahata, T. and Shibata, T. 2010. Evaluation of the antiviral activity of chlorine dioxide and sodium hypochlorite against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. Biocontrol Sci 15(2), 45\u0026ndash;49.\u003c/li\u003e\n \u003cli\u003eSchuster, F.L. and Visvesvara, G.S. 2004. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34(9), 1001\u0026ndash;1027.\u003c/li\u003e\n \u003cli\u003eSiddiqui, R., Ali, I.K.M., Cope, J.R. and Khan, N.A. 2016. Biology and pathogenesis of Naegleria fowleri. Acta Trop 164, 375\u0026ndash;394.\u003c/li\u003e\n \u003cli\u003eSifaoui, I., Rizo-Liendo, A., Reyes-Batlle, M., Arberas-Jim\u0026eacute;nez, I., Rodr\u0026iacute;guez-Exp\u0026oacute;sito, R.L., Pi\u0026ntilde;ero, J.E. and Lorenzo-Morales, J. 2021. Effect of a Commercial Disinfectant CLORICAN\u0026reg; on Acanthamoeba spp. and Naegleria fowleri Viability. Parasitologia 1(3), 119\u0026ndash;129.\u003c/li\u003e\n \u003cli\u003eSohn, H.J., Kim, J.H., Shin, M.H., Song, K.J. and Shin, H.J. 2010. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri. Parasitol Res 106(4), 917\u0026ndash;924.\u003c/li\u003e\n \u003cli\u003eSohn, H.J., Song, K.J., Kang, H., Ham, A.J., Lee, J.H., Chwae, Y.J., Kim, K., Park, S., Kim, J.H. and Shin, H.J. 2019. Cellular characterization of actin gene concerned with contact-dependent mechanisms in Naegleria fowleri. 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Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide. J Appl Microbiol 95(1), 54\u0026ndash;67.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Naegleri fowleri, Acanthamoeba polyphaga, chlorine dioxide (ClO2) gas, disinfectant, amoebicidal effect","lastPublishedDoi":"10.21203/rs.3.rs-3969220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3969220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pathogenic free-living amoebae, \u003cem\u003eNaegleria fowleri\u003c/em\u003e and \u003cem\u003eAcanthamoeba polyphaga\u003c/em\u003e are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO\u003csub\u003e2\u003c/sub\u003e) gas is a potent disinfectant that is harmless to humans. In this study, we examined the amoebicidal effects of ClO\u003csub\u003e2\u003c/sub\u003e gas on \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e. These amoebae were exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e was effectively inhibited by treatment with ClO\u003csub\u003e2\u003c/sub\u003e gas in a time-dependent manner. The growth of \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e exposed to ClO\u003csub\u003e2\u003c/sub\u003e gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective \u003cem\u003eactin\u003c/em\u003e genes were significantly reduced following treatment with ClO\u003csub\u003e2\u003c/sub\u003e gas. ClO\u003csub\u003e2\u003c/sub\u003e gas has an amoebicidal effect on \u003cem\u003eN. fowleri\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e. Therefore, ClO\u003csub\u003e2\u003c/sub\u003e gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.\u003c/p\u003e","manuscriptTitle":"Amoebicidal Effect of Chlorine Dioxide Gas against Pathogenic Naegleria fowleri and Acanthamoeba polyphaga","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 19:23:50","doi":"10.21203/rs.3.rs-3969220/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-27T10:58:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-12T14:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"bc8c9ef4-09c4-4814-9e6c-4eca52568089","date":"2024-03-05T19:31:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-05T15:48:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-23T14:09:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-23T11:48:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2024-02-19T07:04:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c82a9ce5-4bf7-4179-828d-2bd1b620a528","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T22:28:22+00:00","versionOfRecord":{"articleIdentity":"rs-3969220","link":"https://doi.org/10.1007/s00436-024-08215-z","journal":{"identity":"parasitology-research","isVorOnly":false,"title":"Parasitology Research"},"publishedOn":"2024-04-01 22:28:22","publishedOnDateReadable":"April 1st, 2024"},"versionCreatedAt":"2024-02-28 19:23:50","video":"","vorDoi":"10.1007/s00436-024-08215-z","vorDoiUrl":"https://doi.org/10.1007/s00436-024-08215-z","workflowStages":[]},"version":"v1","identity":"rs-3969220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3969220","identity":"rs-3969220","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}