Efficacy of disinfectants and endotoxin retentive filters for the removal of bacterial DNA from dialysates

Efficacy of disinfectants and endotoxin retentive filters for the removal of bacterial DNA from dialysates | 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 Efficacy of disinfectants and endotoxin retentive filters for the removal of bacterial DNA from dialysates Minoru Nakamura, Ami Murata, Toru Yokoyama, Daisuke Furuya, Tomokazu Indo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6124666/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 May, 2025 Read the published version in Renal Replacement Therapy → Version 1 posted You are reading this latest preprint version Abstract Background Bacterial DNA (bDNA) fragments in dialysate lines can trigger inflammatory responses in dialysis patients. However, no studies have reported the removal and inactivation of bDNA in dialysate lines using cleaning and disinfection, and management procedures to control bDNA contamination have yet to be established. Methods The efficiency of an endotoxin retentive filter (ETRF) for the removal of bDNA was examined using an experimental dialysate line incorporating an ETRF and the solubilized materials derived from hot-water-disinfected Pseudomonas aeruginosa cells. To examine the inactivation of bDNA by disinfection, Pseudomonas aeruginosa cell suspensions were disinfected with hot water, peracetic acid, or sodium hypochlorite, and the amount of bDNA remaining after the disinfection treatment was determined. Single-stranded and double-stranded bDNA were measured using Qubit® fluorometry. The molecular size of bDNA was analyzed by polyacrylamide gel electrophoresis. Results In the spike-and-recovery test of solubilized materials derived from hot-water-disinfected bacterial cells, bDNA leakage was observed when the circuit pressure of the inlet ETRF was elevated. bDNA was inactivated more during disinfection with sodium hypochlorite than with peracetic acid and hot water. Conclusions In addition to the ETRF, disinfection with sodium hypochlorite is an effective method for the management of bDNA in dialysates. Bacterial DNA fragments Cleaning and disinfection Dialysate Hot water disinfection Peracetic acid Sodium hypochlorite Sterilization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Ensuring adequate quality control of dialysates is a critical issue in hemodialysis. Good quality dialysates contribute to improvements in the clinical condition of dialysis patients [ 1 , 2 ]. Validation and management of the entire dialysate manufacturing process, including the water treatment system, central dialysis fluid delivery system (CDDS), dialysis machine, and piping for drainage of the dialysate, are necessary for ensuring good dialysate quality. In addition to the international organization for standardization (ISO) [ 3 ], The Japanese Society for Dialysis Therapy and the Japanese Association of Clinical Engineers have proposed many updated guidelines for the management and validation of dialysates [ 4 – 6 ]. Various cleaning and disinfection methods for dialysis lines have been evaluated and validated. One of these disinfection methods uses with water at high temperatures above 80°C. The heat conductivity of the hot water allows for disinfection even in dead spaces where disinfectants cannot access easily. However, its disadvantages are poor organic substance decomposition and high energy costs. Peracetic acid disinfection can remove scales, such as those formed by calcium carbonate, and degrade organic substances [ 7 ]. However, due to its low pH, there are strict regulations regarding wastewater discharge [ 8 ], and the operation of neutralization equipment incurs running costs. Sodium hypochlorite disinfection is very effective in degrading organic substances [ 9 ]; however, it causes metal corrosion, leading to the formation of rust [ 10 ]. Since each conventional disinfection method has its own advantages and disadvantages, the disinfection methods employed differ between facilities. In Japan, disinfection with sodium hypochlorite is commonly practiced because it is inexpensive and recommended by many manufacturers. During the disinfection processes, it is important to control the temperature and concentrations of these agents to effectively remove the biofilm formed by contaminating bacteria in the dialysate line [ 11 – 14 ]. Even if viable bacteria are killed through cleaning and disinfection, endotoxins (ET) and bacterial DNA (bDNA) fragments (bDNAF), a low-molecular-weight oligonucleotide, released from dead bacterial cells can still persist. ET is well known as a potent pyrogen that strongly induces inflammatory responses [ 15 , 16 ]. bDNA is also recognized by the Toll-like receptor, which recognizes pathogen-associated molecular patterns, and causes inflammatory response. bDNAF can enter the bloodstream of hemodialysis patients from the contaminated dialysate passed through the dialysis membrane. Schindler et al. reported that bDNA at a concentration of 500 ng/mL induces the production of approximately 50 pg/mL IL-6 in culture supernatant of human peripheral blood mononuclear cells [ 17 ]. Bossola et al. reported that bDNAF circulating in the bloodstream increases serum CRP and IL-6 levels in hemodialysis patients [ 18 ]. Szeto et al. demonstrated that the bDNAF concentration in blood is a strong predictor of cardiovascular diseases in peritoneal dialysis patients [ 19 ]. In clinical practice, an endotoxin retentive filter (ETRF) is incorporated into the dialysis machine to remove ET and viable bacteria [ 6 ]. However, the pores of hollow-fiber membranes used in ETRFs, such as polysulfone (PS) and polyester polymer alloy (PEPA), can become enlarged after repeated cleaning and disinfection, leading to ET leakage into the dialysate [ 20 , 21 ]. In addition, very small bDNAF can pass through a medium cut-off (MCO) membrane dialyzer [ 22 ]. Based on the above findings, strict management of the dialysate upstream of the ETRF would result in better outcomes. ET and bDNAF contamination of dialysates needs to be controlled to achieve the lowest possible levels of these agents. However, few studies have examined the relationship between bacterial contamination and bDNA levels in dialysates. The guidelines of the ISO, the Japanese Society for Dialysis Therapy, and the Japanese Association for Clinical Engineers make no mention of bDNA contamination in the dialysate [ 3 – 6 ]. Thus, we consider that it is necessary to evaluate bDNA removing and inactivating ability of current cleaning and disinfection procedures and improve the cleanliness of dialysates. It would lead to further improve the quality of life (QOL) of dialysis patients. In this study, we evaluated the bDNA capturing ability of ETRFs and the effectiveness of disinfection processes for bDNA removal. bDNA capture with ETRFs was evaluated by using dialysate spiked with soluble materials derived from hot-water-disinfected Pseudomonas aeruginosa ( P. aeruginosa ) cells. Ability to inactivate bDNA in dialysates of disinfection methods was evaluated by using dialysates spiked with various amounts of P. aeruginosa cells. The ultimate aim of this study is to provide more reliable dialysates, advance dialysis treatment, and improve the QOL of dialysis patients by examining the effectiveness of dialysate management procedures for bDNA contamination during hemodialysis therapy. Methods Quantification of bDNA The amounts of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) were measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA) using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the Qubit ssDNA Assay Kit (Thermo Fisher Scientific), respectively. Evaluation of bDNA removal performance of ETRF The multiple-time-used ETRF [CF-609N precision ultrafiltration filter (Nipro, Osaka, Japan)] was used. Prior to experimental use, the ETRF was enclosed in 800 ppm sodium hypochlorite for 60 min, followed by rinsing with reverse osmosis (RO) water for 60 min. P. aeruginosa ATCC 10145 was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The cells were precultured in 50 mL of Premedia ordinary bouillon medium (Kyokuto Pharmaceutical Industrial, Tokyo, Japan) at 37°C for 24 h. The preculture was centrifuged at 2,200×g for 10 min, and the cell pellet was suspended in 10 mL of physiological saline solution (Otsuka Pharmaceutical, Tokyo, Japan). Hot water disinfection was performed at 99°C for 15 min using a ThermoQ CHB-T2-E dry bath (Hangzho Bioer Technology, Hangzho, China). The treated samples were cooled on ice water for 1 min, and then centrifuged at 13,200×g for 10 min. The supernatant was diluted with RO water to adjust the dsDNA concentration to 74.7 ng/mL (low concentration) and 520 ng/mL (high concentration). Each DNA solution (500 mL) was circulated through the recirculation line for 10 min, and the pump flow rate was adjusted to 650 mL/min. Then the recirculation line was closed and 300 mL of filtrate was collected from post ETRF. The inlet pressure to the ETRF was measured using a PG-100B handy manometer (Nidec Components, Tokyo, Japan). Evaluation of bDNA inactivation after disinfection P.aeruginosa ATCC10145 cells were precultured at 37°C for 24 h in 5 mL of Premedia ordinary bouillon medium. The preculture was centrifuged at 2,200×g for 10 min, and the cell pellet was suspended with 5 mL of physiological saline solution. Bacterial cell suspensions were prepared by diluting the suspensions with physiological saline to achieve 10 1 to 10 8 colony forming units (CFU)/mL. Hot-water disinfection was performed as follows. Bacterial suspension was incubated at 99°C for 15 min using a ThermoQ CHB-T2-E dry bath under condition corresponding an Ao value of 3000 as recommended by ISO 15883 [ 23 ]. After disinfection, the sample was cooled on ice water for 1 min. Peracetic acid disinfection was performed as follows. Peracetic acid-based cleaning agent (Sanacide-NX; Amtec, Osaka, Japan) diluted to 120 ppm (1 mL) was added to 1 mL of bacterial suspension, and incubated for 30 min at room temperature according to the manufacture’s instruction. After disinfection, 4 mL of 0.1 M sodium bicarbonate in the B agent of Kindaly 3E (Fuso Pharmaceutical Industry, Osaka, Japan) was added and incubated at room temperature for 30 min to achieve neutralization. Sodium hypochlorite disinfection was performed as follows. One milliliter of a 12% sodium hypochlorite solution (Nipro, Osaka, Japan) diluted in sterile RO water to 800 ppm was added to 1 mL of bacterial suspension and incubated for 30 min at room temperature in accordance with the manufacture’s instruction. After the sodium hypochlorite disinfection, 1 mL of 1 M Tris-HCl buffer (pH 7.0; Nippon Gene, Tokyo, Japan), which inhibits the DNA-degrading activity of sodium hypochlorite [ 24 ], and incubated for 30 min at room temperature. The resulting disinfected samples were centrifuged at 13,200×g for 10 min, the supernatants were recovered, and the amounts of DNA were measured. The experiments were performed 10 times each. To verify the bactericidal effect of the disinfection procedures, 100 µL of the resulting sample was cultured on nutrient agar (Eiken Chemical, Tokyo, Japan) at 37°C for 48 h. Polyacrylamide electrophoresis of bDNA after disinfection Native polyacrylamide gel electrophoresis (PAGE) was performed according to the Davis’s method [ 25 ] using a MultiGel® II Mini 15/25 13W (Cosmo Bio, Tokyo, Japan) equipped with a DPE-1020 cassette electrophoresis chamber (Cosmo Bio). A 5-bp DNA Ladder (O’RangeRuler; Thermo Fisher Scientific) was used as a molecular size marker. An aliquot (10 µL) of the supernatant after disinfection and neutralization was subjected to electrophoresis at 20 mA for 200 min. The gel was stained using Midori Green Xtra (Nippon Genetics, Tokyo, Japan). Briefly, 10 µL of Midori Green Xtra was added to 100 mL of TAE Buffer (Thermo Fisher Scientific), and the gel was soaked in the solution and shaken for 30 min. DNA was detected at a wavelength of 470 nm using the FAS-Nano Imaging System (Nippon Genetics). Statistical analysis For statistical analysis of bDNA levels, a two-sided Bonferroni test was performed using Pharmaco basic7 (Scientist, Tokyo, Japan). A significance level below 5% was considered to be a statistically significant difference. Results bDNA removal performance by ETRF Table 1 and Fig. 2 show the dsDNA and ssDNA concentrations before and after passing soluble materials derived from hot-water-disinfected P. aeruginosa cells through the ETRF. When the low concentration DNA solution was used, the dsDNA and ssDNA concentrations before the ETRF were 74.7 ± 38.4 ng/mL and 153 ± 49 ng/mL, respectively. After passing through the ETRF, these concentrations decreased below the detection limit (5 ng/mL). When the high concentration DNA solution was used, the dsDNA concentrations were 520 ± 380 ng/mL before the ETRF and 19.7 ± 14.0 ng/mL after the ETRF, representing a significant decrease (p < 0.05)༎The ssDNA concentrations were 1,987 ± 449 ng/mL before the ETRF and 20.5 ± 21.5 ng/mL after the ETRF. ssDNA was removed more effectively than dsDNA (Fig. 2 ). Table 1 dsDNA and ssDNA concentrations in fluids of pre and post-passing through ETRF dose dsDNA (ng/mL) (n = 7) ssDNA (ng/mL) (n = 4) Pre ETRF Post ETRF Pre ETRF Post ETRF High 520 ± 380 19.7 ± 14.0 1987 ± 449 20.5 ± 21.5 Low 74.7 ± 38.4 N.D. 153 ± 49 N.D. N.D.: not detected. The relationship between bDNA concentration after the ETRF and circuit pressure revealed that dsDNA and ssDNA were not detected under circuit pressures of less than 165 mmHg, but were detected at 31.0 ng/mL and 32.0 ng/mL, respectively, at a circuit pressure of 435 mmHg. At a circuit pressure of 510 mmHg, these values were 40.8 ng/mL and 50.0 ng/mL, respectively. The results indicated that bDNA were detected in the final dialysate under elevated circuit pressures (Fig. 3 ). bDNA inactivation by disinfectant treatment Using the 10 8 CFU/mL bacterial suspension, dsDNA at concentrations of 966 ± 154 ng/mL and ssDNA at 3,113 ± 489 ng/mL were detected in soluble materials derived from the hot-water-disinfected cells. After peracetic acid disinfection, dsDNA and ssDNA concentrations slightly decreased to 765 ± 185 ng/mL and 1,190 ± 337 ng/mL, respectively, but the decreases were not statistically significant (Figs. 4 and 5 ). By contrast, after sodium hypochlorite disinfection, dsDNA and ssDNA concentrations decreased to 4 ± 4 ng/mL and 39 ± 27 ng/mL, respectively, an approximately 2-log reduction. Using a 10 7 CFU/mL bacterial suspension, dsDNA concentrations of 330 ± 74 ng/mL and ssDNA concentrations of 640 ± 151 ng/mL were detected after hot water disinfection. After peracetic acid disinfection, the concentrations of dsDNA and ssDNA were significantly reduced to 50 ± 26 ng/mL and 77 ± 45 ng/mL, respectively, whereas those after sodium hypochlorite disinfection were below the detection limit. When 10 6 CFU/mL bacterial suspensions were used, dsDNA and ssDNA concentrations of 1 ± 3 ng/mL and ssDNA at 18 ± 14 ng/mL, respectively, were detected after hot water disinfection, while bDNA were below the detection limit after peracetic acid disinfection and sodium hypochlorite disinfection (Figs. 4 and 5 ). Furthermore, when 10 5 CFU/mL bacterial suspensions or lower were employed, dsDNA and ssDNA concentrations were below the detection limit under all three disinfection conditions. Regardless of the bacterial load, viable bacteria were not detected in any of the samples after disinfection. Molecular size of bDNA after disinfection bDNA were analyzed by PAGE after disinfection (Fig. 6 ). After hot water disinfection, a prominent band was observed at approximate 100 bp, together with ladder bands around it and large amount of a smear in the high molecular weight region. Additionally, the bands less than 100 bp were observed. After peracetic acid disinfection, the intensities of these bands were dramatically reduced; however, the ladder bands above the 100 bp band still remained. After sodium hypochlorite disinfection, no bands were detected. Discussion Because ET and bDNAFs in dialysate can trigger inflammatory responses in dialysis patients, their removal or inactivation is considered extremely important for the prognosis of hemodialysis patients. Although ETRFs are considered an effective method for the removal of ET and bDNAFs, several studies have suggested that they do not completely remove these agents [ 20 , 21 ]. In the present study, we examined bDNA removal efficiency after passing a dialysate spiked with soluble materials obtained from hot-water-disinfected bacterial cells through an experimental circuit (Fig. 1 ). The dialysate spiked with the soluble materials derived from 10 8 bacteria cells contained dsDNA on the order of 10 2 ng/mL and ssDNA on the order of 10 3 ng/mL. When the spiked dialysates were passed through the experimental circuit incorporating an ETRF, bDNA was detected in the filtrate only under elevated circuit pressures. Pressure elevation is likely to occur when the hollow-fiber membrane becomes clogged with large amounts of proteins, nucleic acids, and other organic substances, and the resulting shear stress is likely to fragment the DNA. However, slight pore size expansion due to pressure elevation cannot be ruled out. Further studies of the characteristics of the leaked bDNA, such as its molecular size, are needed. In the present study, bDNA was leaked from ETRF when the column pressure exceeded 400 mmHg (Fig. 3 ). In clinical settings, an alarm of dialysis machine sounds at such high level of pressure, and dialysis will be stopped. It believed that pressure management prevents bDNA leakage. The membrane materials and pore sizes vary depending on the ETRF products, and it provides differences in adsorption capacity and sieving coefficients. The DNA removal performance of different ETRF products should be evaluated in future study. bDNA inactivation through disinfection would be another effective strategy for reducing bDNA in the final dialysate. In the present study, ssDNA levels were higher than dsDNA levels after disinfection, probably because the hydrogen bonds in dsDNA were cleaved by heat or oxidative degradation, producing ssDNA. The highest levels of bDNA were detected in the spiked dialysate after hot water disinfection, suggesting that heat stability of DNA prevents effective bDNA inactivation. Sodium hypochlorite and peracetic acid disinfections were more effective in reducing bDNA levels than hot water disinfection. Sodium hypochlorite disinfection was more effective than peracetic acid disinfection. Notably, neither dsDNA nor ssDNA could be detected after sodium hypochlorite disinfection in dialysates spiked with bacterial cells at a concentration of 10 7 CFU/mL. This indicates that sodium hypochlorite is superior to other disinfection methods for the inactivation of bDNA. However, we have previously reported that prolonged cleaning and disinfection using sodium hypochlorite, which has a strong oxidative effect, leads to metal corrosion and rust formation in dialysis machines [ 10 ]. Furthermore, ferric hydroxide, commonly known as red rust, significantly promotes the growth of P. aeruginosa by promoting biofilm formation. This indicates that inappropriate cleaning and disinfection with sodium hypochlorite can jeopardize the cleanliness of dialysate lines [ 26 ]. Additionally, rust must be removed periodically when sodium hypochlorite is used for extended periods of time to disinfect dialysis lines. [ 27 ]. This study also showed that bDNA was detectable in hot-water-disinfected bacterial suspensions derived from ≥ 10 6 CFU/mL bacterial cells. Cuevas et al. reported that up to 3.8 × 10 7 cells/mL of glucose non-fermenting Gram-negative rods (NFGNR) are present in biofilms on RO membranes [ 28 ], and Ohsono et al. reported that more than 10 7 CFU/mL P. aeruginosa was found in biofilms on dialysis lines [ 14 ]. Biofilms on dialysis lines risk contaminating the final dialysate with high levels of bDNA. In this study, bDNA could not be detected after hot water disinfection of soluble materials derived from containing ≤ 10 5 CFU/mL bacterial cells. The detection limit of the Qubit 4 fluorometer used in this study is 5 ng/mL. However, even bDNA levels below this threshold can potentially result in accumulation of clinically significant amounts of bDNA in the blood of patients during hemodialysis because of the large volume of dialysate (120–200 L) used during this treatment. bDNA at a concentration of 500 ng/mL in blood might induce significant IL-6 production in consideration of the previous report [ 17 ]. Therefore, it is important to maintain bDNA at the lowest possible level. Since DNA is heat-stable, hot water disinfection is unlikely to effectively inactivate bDNA, even with extended disinfection durations. The concentration and disinfection time of disinfectants, such as sodium hypochlorite and peracetic acid, should be optimized for bDNA inactivation to reduce clinical risk. bDNA detected after hot water disinfection included fragmented DNA, namely bDNAF, as observed by PAGE (Fig. 6 ). A major bDNAF band was observed at approximately 100 bp together with high molecular weight DNA. Furthermore, the fragments smaller than 100 bp were also observed. A maximum cutoff value of ETRF is 30,000 Da according to the manufacturer’s documents. The 30,000 Da corresponds to approximately 90 bp for ssDNA and 45 bp for dsDNA. The bDNAF smaller than 100 bp might pass through the ETRF. ETRFs alone cannot completely prevent bDNA contamination in the terminal dialysate. Considering this, novel devices capable of removing or adsorbing bDNA after passage of the dialysate through the ETRF should be developed. However, another solution to develop an effective disinfection program could be to integrate currently available countermeasures. This requires investigating the advantages and disadvantages of each disinfection method. The present study showed that disinfection with sodium hypochlorite was more effective in inactivating bDNA than disinfection with peracetic acid. Conversely, peracetic acid has the advantage of removal of scale, which consists mainly calcium carbonate and forms within dialysate [ 7 ]. The findings in this study suggest that combining hot water disinfection with sodium hypochlorite and/or peracetic acid disinfection might provide an optimal method for keeping bDNA at low levels in dialysate lines. Conclusions bDNA generated by hot water disinfection contains small-sized fragments that could potentially pass through ETRFs. This study shows that sodium hypochlorite disinfection is effective method for inactivating bDNA. The current management of the removal or inactivation of bacterial-derived substances, such as bDNA and endotoxins, in addition to viable bacterial contamination, remains insufficient. Further fundamental investigation is needed for the development of new biological management standards for hemodialysis therapy. Abbreviations bDNA bacterial DNA bDNAF bacterial DNA fragments CFU colony forming unit dsDNA double-stranded DNA ET endotoxin ETRF endotoxin retentive filter ISO international organization for standardization RO reverse osmosis ssDNA single-stranded DNA Declarations Acknowledgements The authors would like to thank members of the Department of Microbiology, Sapporo Medical University School of Medicine (Sapporo, Japan) for their valuable suggestions and discussions. Author Contributions All authors contributed to the conception and design of the study, the critical reading of the article for important intellectual content, and the final approval of the article. Minoru Nakamura and Ami Murata contributed to the collection and assembly of data. Minoru Nakamura, Ami Murata and Shin-ichi Yokota contributed to the analysis and interpretation of the data, and also drafting of the article. Funding None. Availability of data and materials The datasets analyzed during this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Schiffl H, Lang SM, Bergner A. Ultrapure dialysate reduces the dose of recombinant human erythropoietin. Nephron. 1999; 83:278-9. Masakane I. Review: clinical usefulness of ultrapure dialysate: recent evidence and perspectives. 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(in Japanese) Nakamura M, Okayama M, Hagiwara S, Nawa T, Yokota S. Effects of metal corrosion in the pump of a dialysis machine on the sterility of the terminal dialysate by spike-and-recovery testing with bacteria. Renal Replace Ther. 2024; 10:6. Cuevas JP, Morga R, Sánchez-Alonzo K, Valenzuela C, Aguayo P, Smith CT, et al. Characterization of the bacterial biofilm communities present in reverse-osmosis water systems for haemodialysis. Microoganisms. 2020; 8:1418. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 May, 2025 Read the published version in Renal Replacement Therapy → Version 1 posted 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. 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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-6124666","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434993647,"identity":"ca49e253-0fa6-404b-8f22-4c329a95e5eb","order_by":0,"name":"Minoru Nakamura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYFACNhiD+RiMlUCsFrY0krXwmBHnLHP2Y4mfbrbdk2cQO/PtMc8fBnn+BoZnD/BpsexJOyyd21Zs2CCdu92Yt43BcMYBhnQDfFoMDqQDFbclMO6/nbtNmreBgXEDA0OaBF4t5583/wZqsW+QznkmDXSYPWEtN9KOgWxJBGphk+ZhY0gkQsuzNOuccwnJDdJpZpJz2ySSZxwm5Jfzaca3c8oSbBukk59JvPljY9vf3pP2AJ8WdAB0EjNPGmF1aID9GGE1o2AUjIJRMJIAANsEQ4+G1iK6AAAAAElFTkSuQmCC","orcid":"","institution":"Hokkaido University of Science","correspondingAuthor":true,"prefix":"","firstName":"Minoru","middleName":"","lastName":"Nakamura","suffix":""},{"id":434993648,"identity":"6ca90923-2082-4b0b-963e-708b6fef9fe3","order_by":1,"name":"Ami Murata","email":"","orcid":"","institution":"Hokkaido University of Science","correspondingAuthor":false,"prefix":"","firstName":"Ami","middleName":"","lastName":"Murata","suffix":""},{"id":434993649,"identity":"c0a323c7-0e68-4412-9660-0c08a5a4c30b","order_by":2,"name":"Toru Yokoyama","email":"","orcid":"","institution":"Hokkaido University of Science","correspondingAuthor":false,"prefix":"","firstName":"Toru","middleName":"","lastName":"Yokoyama","suffix":""},{"id":434993650,"identity":"13b03ed5-f1d4-4993-af65-6727dda4c0c3","order_by":3,"name":"Daisuke Furuya","email":"","orcid":"","institution":"Hokkaido University of Science","correspondingAuthor":false,"prefix":"","firstName":"Daisuke","middleName":"","lastName":"Furuya","suffix":""},{"id":434993651,"identity":"645fd2ce-afb4-4dfa-a0e3-fd5f6f80505b","order_by":4,"name":"Tomokazu Indo","email":"","orcid":"","institution":"Hokkaido University of Science","correspondingAuthor":false,"prefix":"","firstName":"Tomokazu","middleName":"","lastName":"Indo","suffix":""},{"id":434993652,"identity":"9f799de6-5e3b-491d-b17e-3c94c11bbe4b","order_by":5,"name":"Shin-ichi Yokota","email":"","orcid":"","institution":"Sapporo Medical University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shin-ichi","middleName":"","lastName":"Yokota","suffix":""}],"badges":[],"createdAt":"2025-02-28 02:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6124666/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6124666/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41100-025-00623-w","type":"published","date":"2025-05-09T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79551673,"identity":"0d7e7783-0263-49d0-ab92-5a8c7432d059","added_by":"auto","created_at":"2025-03-31 06:41:42","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228060,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental circuit\u003c/p\u003e\n\u003cp\u003eTo evaluate the bDNA removal performance of the ETRF, samples containing bDNA were introduced into the circuit at a flow rate of 650 mL/min, and samples were taken from before and after the ETRF.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/044390f1d24f61f3eb3c90db.jpeg"},{"id":79551669,"identity":"f6efaf11-0b61-4efc-96f6-72849d32e519","added_by":"auto","created_at":"2025-03-31 06:41:42","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":273031,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of the ETRF for the removal of bDNA from soluble materials derived from hot-water-disinfected \u003cem\u003eP. aeruginosa\u003c/em\u003e cells\u003c/p\u003e\n\u003cp\u003eN.D. (n): not detected (numbers of experiments). Gray plots indicate that bDNA was not detected in dialysis fluids post ETRF. *: p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/75842ae8a4eba21b92cbdbbe.jpeg"},{"id":79552360,"identity":"582e57b5-428d-4a3f-ac2c-bfa605779c0e","added_by":"auto","created_at":"2025-03-31 06:49:42","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171397,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between bDNA concentration and intra-circuit pressure after the ETRF\u003c/p\u003e\n\u003cp\u003eBlack plots indicate that bDNA was not detected in dialysis fluids post ETRF\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/544393f2532e9c69f5755191.jpeg"},{"id":79551671,"identity":"359c66ad-6d25-4391-bd79-0ac9d371e6da","added_by":"auto","created_at":"2025-03-31 06:41:42","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":311440,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of dsDNA concentrations in soluble materials derived from bacterial cells after disinfection with hot water, peracetic acid, or sodium hypochlorite\u003c/p\u003e\n\u003cp\u003eN.D. (n): not detected (numbers of experiments); Heat: hot water disinfection; PA: peracetic acid disinfection; NaClO: sodium hypochlorite disinfection; N.S.: not significant; **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/a34db724b07f67f4b6c41807.jpeg"},{"id":79552358,"identity":"1062e070-5ee9-455c-9df5-eed10ac3000e","added_by":"auto","created_at":"2025-03-31 06:49:42","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":332707,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of ssDNA concentrations in soluble materials derived from bacterial cells after disinfection with hot water, peracetic acid, or sodium hypochlorite\u003c/p\u003e\n\u003cp\u003eN.D. (n): not detected (numbers of experiments); Heat: hot water disinfection; PA: peracetic acid disinfection; NaClO: sodium hypochlorite disinfection; N.S.: not significant; **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/9f515995caac248bd4c8527a.jpeg"},{"id":79551681,"identity":"3155cae7-041e-4b41-a7eb-bd40a812c72c","added_by":"auto","created_at":"2025-03-31 06:41:42","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":825466,"visible":true,"origin":"","legend":"\u003cp\u003ePAGE of bDNA after disinfection\u003c/p\u003e\n\u003cp\u003eNative PAGE was performed on a 15% to 25%(w/v) polyacrylamide gradient gel. The gel was stained with Midori Green Xtra. M: Size marker; 1: hot water disinfection; 2: peracetic acid disinfection; 3: sodium hypochlorite disinfection.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/af92ec2b59f9ac3c2b33cfff.jpeg"},{"id":82537498,"identity":"ad2792d1-d220-4ec8-9e4e-95f0c4c20498","added_by":"auto","created_at":"2025-05-12 16:07:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2782575,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6124666/v1/5b27d861-53d9-4dc8-ae3a-49a10a71a148.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of disinfectants and endotoxin retentive filters for the removal of bacterial DNA from dialysates","fulltext":[{"header":"Background","content":"\u003cp\u003eEnsuring adequate quality control of dialysates is a critical issue in hemodialysis. Good quality dialysates contribute to improvements in the clinical condition of dialysis patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Validation and management of the entire dialysate manufacturing process, including the water treatment system, central dialysis fluid delivery system (CDDS), dialysis machine, and piping for drainage of the dialysate, are necessary for ensuring good dialysate quality. In addition to the international organization for standardization (ISO) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], The Japanese Society for Dialysis Therapy and the Japanese Association of Clinical Engineers have proposed many updated guidelines for the management and validation of dialysates [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious cleaning and disinfection methods for dialysis lines have been evaluated and validated. One of these disinfection methods uses with water at high temperatures above 80\u0026deg;C. The heat conductivity of the hot water allows for disinfection even in dead spaces where disinfectants cannot access easily. However, its disadvantages are poor organic substance decomposition and high energy costs. Peracetic acid disinfection can remove scales, such as those formed by calcium carbonate, and degrade organic substances [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, due to its low pH, there are strict regulations regarding wastewater discharge [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the operation of neutralization equipment incurs running costs. Sodium hypochlorite disinfection is very effective in degrading organic substances [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; however, it causes metal corrosion, leading to the formation of rust [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Since each conventional disinfection method has its own advantages and disadvantages, the disinfection methods employed differ between facilities. In Japan, disinfection with sodium hypochlorite is commonly practiced because it is inexpensive and recommended by many manufacturers. During the disinfection processes, it is important to control the temperature and concentrations of these agents to effectively remove the biofilm formed by contaminating bacteria in the dialysate line [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEven if viable bacteria are killed through cleaning and disinfection, endotoxins (ET) and bacterial DNA (bDNA) fragments (bDNAF), a low-molecular-weight oligonucleotide, released from dead bacterial cells can still persist. ET is well known as a potent pyrogen that strongly induces inflammatory responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. bDNA is also recognized by the Toll-like receptor, which recognizes pathogen-associated molecular patterns, and causes inflammatory response. bDNAF can enter the bloodstream of hemodialysis patients from the contaminated dialysate passed through the dialysis membrane. Schindler et al. reported that bDNA at a concentration of 500 ng/mL induces the production of approximately 50 pg/mL IL-6 in culture supernatant of human peripheral blood mononuclear cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Bossola et al. reported that bDNAF circulating in the bloodstream increases serum CRP and IL-6 levels in hemodialysis patients [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Szeto et al. demonstrated that the bDNAF concentration in blood is a strong predictor of cardiovascular diseases in peritoneal dialysis patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In clinical practice, an endotoxin retentive filter (ETRF) is incorporated into the dialysis machine to remove ET and viable bacteria [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the pores of hollow-fiber membranes used in ETRFs, such as polysulfone (PS) and polyester polymer alloy (PEPA), can become enlarged after repeated cleaning and disinfection, leading to ET leakage into the dialysate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, very small bDNAF can pass through a medium cut-off (MCO) membrane dialyzer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the above findings, strict management of the dialysate upstream of the ETRF would result in better outcomes. ET and bDNAF contamination of dialysates needs to be controlled to achieve the lowest possible levels of these agents. However, few studies have examined the relationship between bacterial contamination and bDNA levels in dialysates. The guidelines of the ISO, the Japanese Society for Dialysis Therapy, and the Japanese Association for Clinical Engineers make no mention of bDNA contamination in the dialysate [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, we consider that it is necessary to evaluate bDNA removing and inactivating ability of current cleaning and disinfection procedures and improve the cleanliness of dialysates. It would lead to further improve the quality of life (QOL) of dialysis patients.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the bDNA capturing ability of ETRFs and the effectiveness of disinfection processes for bDNA removal. bDNA capture with ETRFs was evaluated by using dialysate spiked with soluble materials derived from hot-water-disinfected \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e) cells. Ability to inactivate bDNA in dialysates of disinfection methods was evaluated by using dialysates spiked with various amounts of \u003cem\u003eP. aeruginosa\u003c/em\u003e cells. The ultimate aim of this study is to provide more reliable dialysates, advance dialysis treatment, and improve the QOL of dialysis patients by examining the effectiveness of dialysate management procedures for bDNA contamination during hemodialysis therapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of bDNA\u003c/h2\u003e \u003cp\u003eThe amounts of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) were measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA) using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the Qubit ssDNA Assay Kit (Thermo Fisher Scientific), respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of bDNA removal performance of ETRF\u003c/h3\u003e\n\u003cp\u003eThe multiple-time-used ETRF [CF-609N precision ultrafiltration filter (Nipro, Osaka, Japan)] was used. Prior to experimental use, the ETRF was enclosed in 800 ppm sodium hypochlorite for 60 min, followed by rinsing with reverse osmosis (RO) water for 60 min. \u003cem\u003eP. aeruginosa\u003c/em\u003e ATCC 10145 was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The cells were precultured in 50 mL of Premedia ordinary bouillon medium (Kyokuto Pharmaceutical Industrial, Tokyo, Japan) at 37\u0026deg;C for 24 h. The preculture was centrifuged at 2,200\u0026times;g for 10 min, and the cell pellet was suspended in 10 mL of physiological saline solution (Otsuka Pharmaceutical, Tokyo, Japan). Hot water disinfection was performed at 99\u0026deg;C for 15 min using a ThermoQ CHB-T2-E dry bath (Hangzho Bioer Technology, Hangzho, China). The treated samples were cooled on ice water for 1 min, and then centrifuged at 13,200\u0026times;g for 10 min. The supernatant was diluted with RO water to adjust the dsDNA concentration to 74.7 ng/mL (low concentration) and 520 ng/mL (high concentration). Each DNA solution (500 mL) was circulated through the recirculation line for 10 min, and the pump flow rate was adjusted to 650 mL/min. Then the recirculation line was closed and 300 mL of filtrate was collected from post ETRF. The inlet pressure to the ETRF was measured using a PG-100B handy manometer (Nidec Components, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eEvaluation of bDNA inactivation after disinfection\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eP.aeruginosa\u003c/em\u003e ATCC10145 cells were precultured at 37\u0026deg;C for 24 h in 5 mL of Premedia ordinary bouillon medium. The preculture was centrifuged at 2,200\u0026times;g for 10 min, and the cell pellet was suspended with 5 mL of physiological saline solution. Bacterial cell suspensions were prepared by diluting the suspensions with physiological saline to achieve 10\u003csup\u003e1\u003c/sup\u003e to 10\u003csup\u003e8\u003c/sup\u003e colony forming units (CFU)/mL.\u003c/p\u003e \u003cp\u003eHot-water disinfection was performed as follows. Bacterial suspension was incubated at 99\u0026deg;C for 15 min using a ThermoQ CHB-T2-E dry bath under condition corresponding an Ao value of 3000 as recommended by ISO 15883 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After disinfection, the sample was cooled on ice water for 1 min.\u003c/p\u003e \u003cp\u003ePeracetic acid disinfection was performed as follows. Peracetic acid-based cleaning agent (Sanacide-NX; Amtec, Osaka, Japan) diluted to 120 ppm (1 mL) was added to 1 mL of bacterial suspension, and incubated for 30 min at room temperature according to the manufacture\u0026rsquo;s instruction. After disinfection, 4 mL of 0.1 M sodium bicarbonate in the B agent of Kindaly 3E (Fuso Pharmaceutical Industry, Osaka, Japan) was added and incubated at room temperature for 30 min to achieve neutralization.\u003c/p\u003e \u003cp\u003eSodium hypochlorite disinfection was performed as follows. One milliliter of a 12% sodium hypochlorite solution (Nipro, Osaka, Japan) diluted in sterile RO water to 800 ppm was added to 1 mL of bacterial suspension and incubated for 30 min at room temperature in accordance with the manufacture\u0026rsquo;s instruction. After the sodium hypochlorite disinfection, 1 mL of 1 M Tris-HCl buffer (pH 7.0; Nippon Gene, Tokyo, Japan), which inhibits the DNA-degrading activity of sodium hypochlorite [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and incubated for 30 min at room temperature.\u003c/p\u003e \u003cp\u003eThe resulting disinfected samples were centrifuged at 13,200\u0026times;g for 10 min, the supernatants were recovered, and the amounts of DNA were measured. The experiments were performed 10 times each. To verify the bactericidal effect of the disinfection procedures, 100 \u0026micro;L of the resulting sample was cultured on nutrient agar (Eiken Chemical, Tokyo, Japan) at 37\u0026deg;C for 48 h.\u003c/p\u003e\n\u003ch3\u003ePolyacrylamide electrophoresis of bDNA after disinfection\u003c/h3\u003e\n\u003cp\u003eNative polyacrylamide gel electrophoresis (PAGE) was performed according to the Davis\u0026rsquo;s method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] using a MultiGel\u0026reg; II Mini 15/25 13W (Cosmo Bio, Tokyo, Japan) equipped with a DPE-1020 cassette electrophoresis chamber (Cosmo Bio). A 5-bp DNA Ladder (O\u0026rsquo;RangeRuler; Thermo Fisher Scientific) was used as a molecular size marker. An aliquot (10 \u0026micro;L) of the supernatant after disinfection and neutralization was subjected to electrophoresis at 20 mA for 200 min. The gel was stained using Midori Green Xtra (Nippon Genetics, Tokyo, Japan). Briefly, 10 \u0026micro;L of Midori Green Xtra was added to 100 mL of TAE Buffer (Thermo Fisher Scientific), and the gel was soaked in the solution and shaken for 30 min. DNA was detected at a wavelength of 470 nm using the FAS-Nano Imaging System (Nippon Genetics).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor statistical analysis of bDNA levels, a two-sided Bonferroni test was performed using Pharmaco basic7 (Scientist, Tokyo, Japan). A significance level below 5% was considered to be a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ebDNA removal performance by ETRF\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e show the dsDNA and ssDNA concentrations before and after passing soluble materials derived from hot-water-disinfected \u003cem\u003eP. aeruginosa\u003c/em\u003e cells through the ETRF. When the low concentration DNA solution was used, the dsDNA and ssDNA concentrations before the ETRF were 74.7\u0026thinsp;\u0026plusmn;\u0026thinsp;38.4 ng/mL and 153\u0026thinsp;\u0026plusmn;\u0026thinsp;49 ng/mL, respectively. After passing through the ETRF, these concentrations decreased below the detection limit (5 ng/mL). When the high concentration DNA solution was used, the dsDNA concentrations were 520\u0026thinsp;\u0026plusmn;\u0026thinsp;380 ng/mL before the ETRF and 19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;14.0 ng/mL after the ETRF, representing a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)༎The ssDNA concentrations were 1,987\u0026thinsp;\u0026plusmn;\u0026thinsp;449 ng/mL before the ETRF and 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;21.5 ng/mL after the ETRF. ssDNA was removed more effectively than dsDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003edsDNA and ssDNA concentrations in fluids of pre and post-passing through ETRF\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003edose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003edsDNA (ng/mL) (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003essDNA (ng/mL) (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre ETRF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePost ETRF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePre ETRF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePost ETRF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e520\u0026thinsp;\u0026plusmn;\u0026thinsp;380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;14.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1987\u0026thinsp;\u0026plusmn;\u0026thinsp;449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;21.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e74.7\u0026thinsp;\u0026plusmn;\u0026thinsp;38.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e153\u0026thinsp;\u0026plusmn;\u0026thinsp;49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eN.D.: not detected.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relationship between bDNA concentration after the ETRF and circuit pressure revealed that dsDNA and ssDNA were not detected under circuit pressures of less than 165 mmHg, but were detected at 31.0 ng/mL and 32.0 ng/mL, respectively, at a circuit pressure of 435 mmHg. At a circuit pressure of 510 mmHg, these values were 40.8 ng/mL and 50.0 ng/mL, respectively. The results indicated that bDNA were detected in the final dialysate under elevated circuit pressures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ebDNA inactivation by disinfectant treatment\u003c/h3\u003e\n\u003cp\u003eUsing the 10\u003csup\u003e8\u003c/sup\u003e CFU/mL bacterial suspension, dsDNA at concentrations of 966\u0026thinsp;\u0026plusmn;\u0026thinsp;154 ng/mL and ssDNA at 3,113\u0026thinsp;\u0026plusmn;\u0026thinsp;489 ng/mL were detected in soluble materials derived from the hot-water-disinfected cells. After peracetic acid disinfection, dsDNA and ssDNA concentrations slightly decreased to 765\u0026thinsp;\u0026plusmn;\u0026thinsp;185 ng/mL and 1,190\u0026thinsp;\u0026plusmn;\u0026thinsp;337 ng/mL, respectively, but the decreases were not statistically significant (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). By contrast, after sodium hypochlorite disinfection, dsDNA and ssDNA concentrations decreased to 4\u0026thinsp;\u0026plusmn;\u0026thinsp;4 ng/mL and 39\u0026thinsp;\u0026plusmn;\u0026thinsp;27 ng/mL, respectively, an approximately 2-log reduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing a 10\u003csup\u003e7\u003c/sup\u003e CFU/mL bacterial suspension, dsDNA concentrations of 330\u0026thinsp;\u0026plusmn;\u0026thinsp;74 ng/mL and ssDNA concentrations of 640\u0026thinsp;\u0026plusmn;\u0026thinsp;151 ng/mL were detected after hot water disinfection. After peracetic acid disinfection, the concentrations of dsDNA and ssDNA were significantly reduced to 50\u0026thinsp;\u0026plusmn;\u0026thinsp;26 ng/mL and 77\u0026thinsp;\u0026plusmn;\u0026thinsp;45 ng/mL, respectively, whereas those after sodium hypochlorite disinfection were below the detection limit.\u003c/p\u003e \u003cp\u003eWhen 10\u003csup\u003e6\u003c/sup\u003e CFU/mL bacterial suspensions were used, dsDNA and ssDNA concentrations of 1\u0026thinsp;\u0026plusmn;\u0026thinsp;3 ng/mL and ssDNA at 18\u0026thinsp;\u0026plusmn;\u0026thinsp;14 ng/mL, respectively, were detected after hot water disinfection, while bDNA were below the detection limit after peracetic acid disinfection and sodium hypochlorite disinfection (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, when 10\u003csup\u003e5\u003c/sup\u003e CFU/mL bacterial suspensions or lower were employed, dsDNA and ssDNA concentrations were below the detection limit under all three disinfection conditions.\u003c/p\u003e \u003cp\u003eRegardless of the bacterial load, viable bacteria were not detected in any of the samples after disinfection.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMolecular size of bDNA after disinfection\u003c/h2\u003e \u003cp\u003ebDNA were analyzed by PAGE after disinfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After hot water disinfection, a prominent band was observed at approximate 100 bp, together with ladder bands around it and large amount of a smear in the high molecular weight region. Additionally, the bands less than 100 bp were observed. After peracetic acid disinfection, the intensities of these bands were dramatically reduced; however, the ladder bands above the 100 bp band still remained. After sodium hypochlorite disinfection, no bands were detected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBecause ET and bDNAFs in dialysate can trigger inflammatory responses in dialysis patients, their removal or inactivation is considered extremely important for the prognosis of hemodialysis patients. Although ETRFs are considered an effective method for the removal of ET and bDNAFs, several studies have suggested that they do not completely remove these agents [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, we examined bDNA removal efficiency after passing a dialysate spiked with soluble materials obtained from hot-water-disinfected bacterial cells through an experimental circuit (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The dialysate spiked with the soluble materials derived from 10\u003csup\u003e8\u003c/sup\u003e bacteria cells contained dsDNA on the order of 10\u003csup\u003e2\u003c/sup\u003e ng/mL and ssDNA on the order of 10\u003csup\u003e3\u003c/sup\u003e ng/mL. When the spiked dialysates were passed through the experimental circuit incorporating an ETRF, bDNA was detected in the filtrate only under elevated circuit pressures. Pressure elevation is likely to occur when the hollow-fiber membrane becomes clogged with large amounts of proteins, nucleic acids, and other organic substances, and the resulting shear stress is likely to fragment the DNA. However, slight pore size expansion due to pressure elevation cannot be ruled out. Further studies of the characteristics of the leaked bDNA, such as its molecular size, are needed. In the present study, bDNA was leaked from ETRF when the column pressure exceeded 400 mmHg (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In clinical settings, an alarm of dialysis machine sounds at such high level of pressure, and dialysis will be stopped. It believed that pressure management prevents bDNA leakage. The membrane materials and pore sizes vary depending on the ETRF products, and it provides differences in adsorption capacity and sieving coefficients. The DNA removal performance of different ETRF products should be evaluated in future study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ebDNA inactivation through disinfection would be another effective strategy for reducing bDNA in the final dialysate. In the present study, ssDNA levels were higher than dsDNA levels after disinfection, probably because the hydrogen bonds in dsDNA were cleaved by heat or oxidative degradation, producing ssDNA. The highest levels of bDNA were detected in the spiked dialysate after hot water disinfection, suggesting that heat stability of DNA prevents effective bDNA inactivation. Sodium hypochlorite and peracetic acid disinfections were more effective in reducing bDNA levels than hot water disinfection. Sodium hypochlorite disinfection was more effective than peracetic acid disinfection. Notably, neither dsDNA nor ssDNA could be detected after sodium hypochlorite disinfection in dialysates spiked with bacterial cells at a concentration of 10\u003csup\u003e7\u003c/sup\u003e CFU/mL. This indicates that sodium hypochlorite is superior to other disinfection methods for the inactivation of bDNA.\u003c/p\u003e \u003cp\u003eHowever, we have previously reported that prolonged cleaning and disinfection using sodium hypochlorite, which has a strong oxidative effect, leads to metal corrosion and rust formation in dialysis machines [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, ferric hydroxide, commonly known as red rust, significantly promotes the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e by promoting biofilm formation. This indicates that inappropriate cleaning and disinfection with sodium hypochlorite can jeopardize the cleanliness of dialysate lines [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, rust must be removed periodically when sodium hypochlorite is used for extended periods of time to disinfect dialysis lines. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study also showed that bDNA was detectable in hot-water-disinfected bacterial suspensions derived from \u0026ge;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL bacterial cells. Cuevas et al. reported that up to 3.8 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mL of glucose non-fermenting Gram-negative rods (NFGNR) are present in biofilms on RO membranes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and Ohsono et al. reported that more than 10\u003csup\u003e7\u003c/sup\u003e CFU/mL \u003cem\u003eP. aeruginosa\u003c/em\u003e was found in biofilms on dialysis lines [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Biofilms on dialysis lines risk contaminating the final dialysate with high levels of bDNA. In this study, bDNA could not be detected after hot water disinfection of soluble materials derived from containing\u0026thinsp;\u0026le;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e CFU/mL bacterial cells. The detection limit of the Qubit 4 fluorometer used in this study is 5 ng/mL. However, even bDNA levels below this threshold can potentially result in accumulation of clinically significant amounts of bDNA in the blood of patients during hemodialysis because of the large volume of dialysate (120\u0026ndash;200 L) used during this treatment. bDNA at a concentration of 500 ng/mL in blood might induce significant IL-6 production in consideration of the previous report [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, it is important to maintain bDNA at the lowest possible level. Since DNA is heat-stable, hot water disinfection is unlikely to effectively inactivate bDNA, even with extended disinfection durations. The concentration and disinfection time of disinfectants, such as sodium hypochlorite and peracetic acid, should be optimized for bDNA inactivation to reduce clinical risk.\u003c/p\u003e \u003cp\u003ebDNA detected after hot water disinfection included fragmented DNA, namely bDNAF, as observed by PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). A major bDNAF band was observed at approximately 100 bp together with high molecular weight DNA. Furthermore, the fragments smaller than 100 bp were also observed. A maximum cutoff value of ETRF is 30,000 Da according to the manufacturer\u0026rsquo;s documents. The 30,000 Da corresponds to approximately 90 bp for ssDNA and 45 bp for dsDNA. The bDNAF smaller than 100 bp might pass through the ETRF. ETRFs alone cannot completely prevent bDNA contamination in the terminal dialysate. Considering this, novel devices capable of removing or adsorbing bDNA after passage of the dialysate through the ETRF should be developed. However, another solution to develop an effective disinfection program could be to integrate currently available countermeasures. This requires investigating the advantages and disadvantages of each disinfection method. The present study showed that disinfection with sodium hypochlorite was more effective in inactivating bDNA than disinfection with peracetic acid. Conversely, peracetic acid has the advantage of removal of scale, which consists mainly calcium carbonate and forms within dialysate [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The findings in this study suggest that combining hot water disinfection with sodium hypochlorite and/or peracetic acid disinfection might provide an optimal method for keeping bDNA at low levels in dialysate lines.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ebDNA generated by hot water disinfection contains small-sized fragments that could potentially pass through ETRFs. This study shows that sodium hypochlorite disinfection is effective method for inactivating bDNA. The current management of the removal or inactivation of bacterial-derived substances, such as bDNA and endotoxins, in addition to viable bacterial contamination, remains insufficient. Further fundamental investigation is needed for the development of new biological management standards for hemodialysis therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ebDNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;bacterial DNA\u003c/p\u003e\n\u003cp\u003ebDNAF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;bacterial DNA fragments\u003c/p\u003e\n\u003cp\u003eCFU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;colony forming unit\u003c/p\u003e\n\u003cp\u003edsDNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;double-stranded DNA\u003c/p\u003e\n\u003cp\u003eET\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;endotoxin\u003c/p\u003e\n\u003cp\u003eETRF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;endotoxin retentive filter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eISO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;international organization for standardization\u003c/p\u003e\n\u003cp\u003eRO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;reverse osmosis\u003c/p\u003e\n\u003cp\u003essDNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;single-stranded DNA\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank members of the Department of Microbiology, Sapporo Medical University School of Medicine (Sapporo, Japan) for their valuable suggestions and discussions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study, the critical reading of the article for important intellectual content, and the final approval of the article. Minoru Nakamura and Ami Murata contributed to the collection and assembly of data. Minoru Nakamura, Ami Murata and Shin-ichi Yokota contributed to the analysis and interpretation of the data, and also drafting of the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchiffl H, Lang SM, Bergner A. Ultrapure dialysate reduces the dose of recombinant human erythropoietin. Nephron. 1999; 83:278-9.\u003c/li\u003e\n\u003cli\u003eMasakane I. Review: clinical usefulness of ultrapure dialysate: recent evidence and perspectives. Ther Apher Dial. 2006; 10:348-54.\u003c/li\u003e\n\u003cli\u003eISO 23500-5:2019(en). Preparation and quality management of fluids for hemodialysis and related therapies Part 5: Quality of dialysis fluid for hemodialysis and related therapies. 2019. https://www.iso.org/obp/ui/#iso:std:iso:23500:-5:ed-1:v1:en. Accessed 27 Feb 2025.\u003c/li\u003e\n\u003cli\u003eKawanishi H, Akiba T, Masakane I, Tomo T, Mineshima M, Kawasaki T, et al. Standard on microbiological management of fluids for hemodialysis and related therapies by the Japanese Society for Dialysis Therapy 2008. Ther Apher Dial 2009; 13:161\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eMineshima M, Kawanishi H, Ase T, Kawasaki T, Tomo T, Nakamoto H, et al. 2016 update Japanese Society for Dialysis Therapy Standard of fluids for hemodialysis and related therapies. Ren Replace Ther. 2018; 4:15.\u003c/li\u003e\n\u003cli\u003eMasakane I, Kawanishi H, Mineshima M, Takemoto Y, Uchino J, Hoshino T, et al. 2011 JSDT Standard on the management of endotoxin retentive filter for dialysis and related therapies. Ther Apher Dial. 2013; 17:229\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eKato M, Sugiura M, Kamiya E, Sakurai H, Nii N, Miyamoto A, et al. The germicidal and cleansing effects of peracetic acid on hemodialysis equipment. J Jpn Soc Dial Ther. 1996; 29:1495-501. (in Japanese)\u003c/li\u003e\n\u003cli\u003eJapan Association for Clinical Engineers. 2019 dialysis wastewater standards compliance procedure, Manual Ver 1.00. 2020. https://ja-ces.or.jp/wordpress/wp-content/uploads/2020/04/fdc2bdc425a56b7db5f6def450e69b52.pdf. Accessed 27 Feb 2025. (in Japanese)\u003c/li\u003e\n\u003cli\u003eEstrela C, Estrela CR, Barbin EL, Spano JC, Marchesan MA, Pecora JD. Mechanism of action of sodium hypochlorite. Braz Dent J. 2002; 13:113-7.\u003c/li\u003e\n\u003cli\u003eNakamura M, Okayama M, Kimura K, Shibata H, Hagiwara M, Nawa T, et al. Isolation several bacteria from the corrosion confirmed pump parts in the bedside console for hemodialysis therapy. J Jpn Assoc Clin Eng. 2017; 61:109-15. (in Japanese)\u003c/li\u003e\n\u003cli\u003eSakuma K, Uchiumi N, Sato S, Aida N, Ishimatsu T, Igoshi T, et al. Experience of using heat citric acid disinfection method in central dialysis fluid delivery system. J Artif Organs. 2010; 13: 145-50.\u003c/li\u003e\n\u003cli\u003eIsakozawa Y, H. Migita, S. Takesawa. Efficacy of biofilm removal from hemodialysis piping. Nephrourol Mon, 2016; 8: e39332.\u003c/li\u003e\n\u003cli\u003eTagaya M, Oda Y, Kimura A, Irifune R, Okano S, Muraktaka T, et al. An easy disinfection strategy for pipes connecting hemodialysis equipment. Int J Artif Organs, 2021; 44: 385-92.\u003c/li\u003e\n\u003cli\u003eOsono E, Honda K, Inoue Y, Ichimura K, Kamano C, Akimoto T, et al. Sodium hypochlorite is effective against biofilms in dialysis equipment. Biocontrol Sci, 2021; 26:1-7.\u003c/li\u003e\n\u003cli\u003eMichie HR, Manogue KR, Spriggs DR, Revhaug A, O\u0026apos;Dwyer S, Dinarello CA, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med, 1988; 318: 1481-6.\u003c/li\u003e\n\u003cli\u003eGiovanni P, Giuseppe G, Loreto G, Francesco PS. Clinical relevance of production in hemodialysis. Kidney Int, 2000; 58, Suppl. 76: S104-11.\u003c/li\u003e\n\u003cli\u003eSchindler R, Beck W, Deppisch R, Aussieker M, Wilde A, G\u0026ouml;hl H, et.al. Short bacterial DNA fragments: detection in dialysate and induction of cytokines. J Am Soc Nephrol. 2004; 15:3207-14.\u003c/li\u003e\n\u003cli\u003eBossola M, Sansuinetti M, Scribano D, Zuppi C, Giungi S, Luciani G, et.al. Circulating bacterial-derived DNA fragments and markers of inflammation in chronic hemodialysis patients. Clin J Am Soc Nephrol. 2009; 4:379-85.\u003c/li\u003e\n\u003cli\u003eSzeto C-C, Kwan BC-H, Chow K-M, Kwok JS-S, Lai K-B, Cheng PM-S, et.al. Circulating bacterial-derived DNA fragment level is a strong predictor of cardiovascular disease in peritoneal dialysis patients. PLoS One. 2015; 10:e0125162.\u003c/li\u003e\n\u003cli\u003eSchepers E, Glorieux G, Eloot S, Hulko M, Boschetti-de-Fierro A, Beck W, et al．Assessment of the association between increasing membrane pore size and endotoxin permeability using a novel experimental dialysis simulation set-up. BMC Nephrol. 2018; 19:1.\u003c/li\u003e\n\u003cli\u003eNozaki H, Tange Y, Inade Y, Uchino T, Azama N. Leakage of endotoxins through the endotoxin retentive filter: An \u003cem\u003ein vitro\u003c/em\u003e study. Blood Purif. 2022; 51:831-9.\u003c/li\u003e\n\u003cli\u003eHulko M, Dietrich V, Koch I, Gekeler A, Gebwert M, Beck W, et al. Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyzer membranes. Sci Rep. 2019; 9:6791. \u003c/li\u003e\n\u003cli\u003eISO 15883-1:2024(en). Washer-disinfectors Part 1: General requirements, terms and definitions and tests. 2024. https://www.iso.org/standard/81249.html. Accessed 21 Mar 2025.\u003c/li\u003e\n\u003cli\u003eShirakawa S, Nishiyama K. Removal of contamination during PCR: application of sodium hypochlorite. J Clin Lab Med. 1992; 36 (12):1265-9. (in Japanese)\u003c/li\u003e\n\u003cli\u003eDavis BJ. Disc Electrophoresis. II. Method and application to human serum proteins. Ann N Y Acad Sci. 1964; 121:404-27.\u003c/li\u003e\n\u003cli\u003eNakamura M, Okayama M, Kimura K, Shibata H, Hagiwara S, Nawa T, et al. Influence of metal pump corrosion on the contamination of terminal dialysis fluid by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. J Jpn Soc Dial Ther. 2019; 52:7-13. (in Japanese)\u003c/li\u003e\n\u003cli\u003eNakamura M, Okayama M, Hagiwara S, Nawa T, Yokota S. Effects of metal corrosion in the pump of a dialysis machine on the sterility of the terminal dialysate by spike-and-recovery testing with bacteria. Renal Replace Ther. 2024; 10:6.\u003c/li\u003e\n\u003cli\u003eCuevas JP, Morga R, S\u0026aacute;nchez-Alonzo K, Valenzuela C, Aguayo P, Smith CT, et al. Characterization of the bacterial biofilm communities present in reverse-osmosis water systems for haemodialysis. Microoganisms. 2020; 8:1418.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacterial DNA fragments, Cleaning and disinfection, Dialysate, Hot water disinfection, Peracetic acid, Sodium hypochlorite, Sterilization","lastPublishedDoi":"10.21203/rs.3.rs-6124666/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6124666/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBacterial DNA (bDNA) fragments in dialysate lines can trigger inflammatory responses in dialysis patients. However, no studies have reported the removal and inactivation of bDNA in dialysate lines using cleaning and disinfection, and management procedures to control bDNA contamination have yet to be established.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe efficiency of an endotoxin retentive filter (ETRF) for the removal of bDNA was examined using an experimental dialysate line incorporating an ETRF and the solubilized materials derived from hot-water-disinfected \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e cells. To examine the inactivation of bDNA by disinfection, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e cell suspensions were disinfected with hot water, peracetic acid, or sodium hypochlorite, and the amount of bDNA remaining after the disinfection treatment was determined. Single-stranded and double-stranded bDNA were measured using Qubit\u0026reg; fluorometry. The molecular size of bDNA was analyzed by polyacrylamide gel electrophoresis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the spike-and-recovery test of solubilized materials derived from hot-water-disinfected bacterial cells, bDNA leakage was observed when the circuit pressure of the inlet ETRF was elevated. bDNA was inactivated more during disinfection with sodium hypochlorite than with peracetic acid and hot water.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn addition to the ETRF, disinfection with sodium hypochlorite is an effective method for the management of bDNA in dialysates.\u003c/p\u003e","manuscriptTitle":"Efficacy of disinfectants and endotoxin retentive filters for the removal of bacterial DNA from dialysates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 06:41:38","doi":"10.21203/rs.3.rs-6124666/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba32f285-6c31-4894-bb61-b84d4adc0518","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:01:38+00:00","versionOfRecord":{"articleIdentity":"rs-6124666","link":"https://doi.org/10.1186/s41100-025-00623-w","journal":{"identity":"renal-replacement-therapy","isVorOnly":false,"title":"Renal Replacement Therapy"},"publishedOn":"2025-05-09 15:57:28","publishedOnDateReadable":"May 9th, 2025"},"versionCreatedAt":"2025-03-31 06:41:38","video":"","vorDoi":"10.1186/s41100-025-00623-w","vorDoiUrl":"https://doi.org/10.1186/s41100-025-00623-w","workflowStages":[]},"version":"v1","identity":"rs-6124666","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6124666","identity":"rs-6124666","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}