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

Renal Replacement Therapy volume 11, Article number: 28 (2025) Cite this article

Abstract

Background

Bacterial DNA (bDNA) fragments in dialysate lines can trigger inflammatory responses in patients on dialysis. 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, P. 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.

Background

Ensuring adequate quality control of dialysates is a critical issue in hemodialysis. Good-quality dialysates contribute to improvements in the clinical condition of patients on dialysis [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 [4,5,6].

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 [11,12,13,14].

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 patients on hemodialysis through 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 patients on hemodialysis [18]. Szeto et al. demonstrated that the bDNAF concentration in blood is a strong predictor of cardiovascular diseases in patients on peritoneal dialysis [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 cutoff (MCO) membrane dialyzer [22].

On the basis of 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 [3,4,5,6]. Thus, the authors consider it necessary to evaluate the ability of current cleaning and disinfection procedures to remove and inactivate bDNA and improve the cleanliness of dialysates. This would lead to further improve the quality of life (QOL) of patients on dialysis.

In this study, the authors 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 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 patients on dialysis 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 the bDNA removal performance of ETRF

ETRF [CF-609N precision ultrafiltration filter (Nipro, Osaka, Japan)] that had been used multiple times was used. The EFTR was incorporated into the experimental circuit (Fig. 1). 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 2200 ×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 using 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 post-ETRF. The inlet pressure to the ETRF was measured using a PG-100B handy manometer (Nidec Components, Tokyo, Japan).

Fig. 1
figure 1

Experimental circuit. To 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

Full size image

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 2200 ×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 101–108 colony-forming units (CFU) per 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 conditions corresponding to an A0 value of 3000, as recommended by ISO 15883 [23]. An A0 value of 3000 is specified as the minimum requirement for heat sterilization, with a holding time of 3000 s (50 min) at 80 °C. After disinfection, the sample was cooled using 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. First, 1 mL 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], was added 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 ten 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 gel 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 Tris-acetate-ethylenediaminetetraacetic acid (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 present 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 1987 ± 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 before and after passing through ETRF
Full size table
Fig. 2
figure 2

Performance of the ETRF when it came to the removal of bDNA from soluble materials derived from hot-water-disinfected P. aeruginosa cells. Gray plots indicate that bDNA was not detected in dialysis fluids post ETRF. ND (n), not detected (number of experiments). *p < 0.05

Full size image

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).

Fig. 3
figure 3

Relationship between bDNA concentration and intra-circuit pressure after the ETRF. Black plots indicate that bDNA was not detected in dialysis fluids following ETRF

Full size image

bDNA inactivation by disinfectant treatment

Using the 108 CFU/mL bacterial suspension, dsDNA at concentrations of 966 ± 154 ng/mL and ssDNA at 3113 ± 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 1190 ± 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.

Fig. 4
figure 4

Comparison of dsDNA concentrations in soluble materials derived from bacterial cells after disinfection with hot water, peracetic acid, or sodium hypochlorite. ND (n), not detected (number of experiments); Heat, hot water disinfection; PA, peracetic acid disinfection; NaClO, sodium hypochlorite disinfection; NS, not significant. **p < 0.01

Full size image
Fig. 5
figure 5

Comparison of ssDNA concentrations in soluble materials derived from bacterial cells after disinfection with hot water, peracetic acid, or sodium hypochlorite. ND (n), not detected (number of experiments); Heat, hot water disinfection; PA, peracetic acid disinfection; NaClO, sodium hypochlorite disinfection; NS, not significant. **p < 0.01

Full size image

Using a 107 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 106 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, 5). Furthermore, when 105 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.

Fig. 6
figure 6

PAGE of bDNA after disinfection. Native PAGE was performed on a 15–25% (w/v) polyacrylamide gradient gel. The gel was stained with Midori Green Xtra. M is a size marker, and 1 is hot water disinfection, 2 peracetic acid disinfection, and 3 sodium hypochlorite disinfection

Full size image

Discussion

Because ET and bDNAFs in dialysate can trigger inflammatory responses in patients on dialysis, their removal or inactivation is considered extremely important for the prognosis of patients on hemodialysis. 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, the authors 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 108 bacteria cells contained dsDNA on the order of 102 ng/mL and ssDNA on the order of 103 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 on the dialysis machine sounds at such a high level of pressure, and dialysis would be stopped. It believed that pressure management prevents bDNA leakage. The membrane materials and pore sizes vary depending on the ETRF product, and this 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 the 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 107 CFU/mL. This indicates that sodium hypochlorite is superior to other disinfection methods for the inactivation of bDNA.

However, the authors 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 ≥ 106 CFU/mL bacterial cells. Cuevas et al. reported that up to 3.8 × 107 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 107 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 ≤ 105 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 dialysis line [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 an 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.

Availability of data and materials

The datasets analyzed during this study are available from the corresponding author upon reasonable request.

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

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Acknowledgements

The authors would like to thank members of the Department of Microbiology at Sapporo Medical University School of Medicine (Sapporo, Japan) for their valuable suggestions and discussions.

Funding

None.

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Authors and Affiliations

  1. Department of Clinical Engineering, Faculty of Health Sciences, Hokkaido University of Science, Sapporo, Japan

    Minoru Nakamura

  2. Department of Medical Technology, Graduate School of Health Sciences, Hokkaido University of Science, Sapporo, Japan

    Ami Murata, Toru Yokoyama, Daisuke Furuya & Tomokazu Indo

  3. Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan

    Shin-ichi Yokota

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  4. Daisuke Furuya
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Contributions

Minoru Nakamura, Ami Murata, Toru Yokoyama, Daisuke Furuya, Tomokazu Indo, and Shin-ichi Yokota 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.

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Correspondence to Minoru Nakamura.

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Nakamura, M., Murata, A., Yokoyama, T. et al. Efficacy of disinfectants and endotoxin-retentive filters for the removal of bacterial DNA from dialysates. Ren Replace Ther 11, 28 (2025). https://doi.org/10.1186/s41100-025-00623-w

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  • DOI: https://doi.org/10.1186/s41100-025-00623-w

Keywords

Renal Replacement Therapy

ISSN: 2059-1381

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