Microbiological survey of dialysate: vantage of use of sterile bag concentrate

Haemodialysed patients are exposed to nearly 400 litres of dialysis water weekly. The bacterial contamination of treated dialysate and water induces acute pyrogenic reactions or chronic damage and cytokine activation. The aim of this study was to value the microbiological parameters of dialysis water and dialysate of our monitors by bacterial culture (measured as colony forming units [CFU]) of water samples at 37 degrees C after 48 hours, at 22 degrees C after 72 hours and after seven days, and by measuring endotoxin levels (endotoxin units [EU]). In our centre, there are 16 monitors (6 monitors use sterile dialysate fluid and 10 monitors use non sterile dialysate fluid). The chemicals used for disinfection are chlorine and paracetic acid. Water samples were taken under sterile procedures every three months for a year. No bacteria were found in the samples of water of the dialysis ring; EU were lower than the limit value of 0.25 EU/ml fixed by the European Pharmacopoeia. The concentration of CFU and EU of the dialysate, taken from monitors with a sterile bag, were lower than those of other monitors (p < 0.05 t Student test). However, the levels of CFU/ml and EU/ml of dialysate samples, taken from monitors with a non-sterile bag, were lower than the guideline value of the European Pharmacopoeia (v.n. CFU < 50 CFU/ml and EU < 0.05 EU/ml). Frequent examination of CFU and EU is essential to reduce the damage caused by the use of contaminated water, therefore the goal of future dialytic techniques will be the use of "sterile dialysate".     

Ultrapure dialysate reduces plasma levels of beta2-microglobulin and pentosidine in hemodialysis patients

BACKGROUND: beta2-Microglobulin (beta2MG) and carbonyl stress are reported to contribute to the development of dialysis-related amyloidosis. The aim of this study was to determine whether the purity of dialysate affects plasma levels of beta2MG and pentosidine (a surrogate marker of carbonyl stress) in hemodialysis patients. METHODS: Sixteen patients on hemodialysis with a polysulfone membrane participated in this study. We switched the dialysate from conventional dialysate (endotoxin level 0.055-0.066 endotoxin units (EU)/ml) to ultrapure dialysate (endotoxin level <0.001 EU/ml), followed patients for 6 months, and then switched back to conventional dialysate once again. Plasma levels of beta2MG, pentosidine, CRP and interleukin-6 (IL-6) were determined before the switch to ultrapure dialysate, 1 and 6 months after the switch to ultrapure dialysate, and 1 month after the switch back to conventional dialysate. RESULTS: The switch from conventional to ultrapure dialysate significantly decreased plasma levels of beta2MG, from 30.1 +/- 1.4 to 27.1 +/- 1.4 mg/dl (p < 0.05) and pentosidine, from 1,535.8 +/- 107.5 to 1,267.6 +/- 102.9 nmol/l (p < 0.01) after 1 month of use. The change of dialysate also significantly decreased plasma levels of CRP, from 0.28 +/- 0.09 to 0.14 +/- 0.05 mg/dl (p < 0.05) and IL-6, from 9.4 +/- 2.7 to 3.5 +/- 0.8 pg/ml (p < 0.01) over the 1-month period. These changes in plasma levels of beta2MG, pentosidine, CRP and IL-6 were maintained over 6 months after switching to ultrapure dialysate and returned to basal levels by switching back to a conventional dialysate. CONCLUSIONS: Ultrapure dialysate decreases plasma levels of beta2MG, pentosidine and inflammatory markers in hemodialysis patients. The use of ultrapure dialysate might be useful in preventing and/or treating complications of dialysis, such as dialysis-related amyloidosis, atherosclerosis and malnutrition.     

透析用水及透析液中内毒素污染状况的分析

目的　研究透析用水、透析液内毒素及细菌污染状况。方法　对北京１８ 家医院透析室反渗水、透析液用改良鲎试验检测内毒素,用血琼脂培养基做细菌培养,用ＥＬＩＳＡ法测定患者血浆白介素（ＩＬ）１、ＩＬ６ 及肿瘤坏死因子（ＴＮＦ）α。结果　反渗水内毒素为（０．１１６±０ ．０６３） ＥＵ／ｍｌ;细菌培养有两家医院阳性,均为１００ ＣＦＵ／ｍｌ。Ｂ浓缩透析液内毒素为（０．４６ ±０．３５） ＥＵ／ｍｌ,而Ａ浓缩透析液内毒素仅（０．０９１±０ ．０８４） ＥＵ／ｍｌ（Ｐ＜０ ．００１） ;Ｂ浓缩透析液细菌培养１１ 家阳性,其中８ 家≥２ ０００ ＣＦＵ／ｍｌ,Ａ浓缩透析液细菌培养阳性仅３ 家,均＜ ２ ０００ ＣＦＵ／ｍｌ。１６ 例患者中有５ 例透析器入口透析液内毒素＞０．５ ＥＵ／ｍｌ,且透后血ＩＬ６ 及ＴＮＦα显著增高。结论　目前透析用水及透析液存在内毒素及细菌的污染,定期检测反渗水、透析液的内毒素水平及进行细菌培养,定期消毒反渗水装置及透析液容器,对减少热源反应至关重要。     

'Clean dialysate' requires not only lower levels of endotoxin but also sterility of dilution water

Clean dialysate should be used in dialysis with a high-flux dialysis membrane to avoid contamination of endotoxin into blood circuits. For this purpose, we should not only clean up the end stream dialysate by endotoxin-cut filters, but also prevent bacterial growth in dilution water lines. Delivery lines of the prepared dialysate from a central dialysate-supplying machine are sterilized in all dialysis facilities, but those of the dilution water in cases of using personal dialysis machines are not sterilized in most facilities in Japan. In our dialysis department, the endotoxin level in the dilution water lines decreased to <50 EU/l in 3 weeks by sterilization with a low concentration of sodium hypochlorite (30-100 ppm) once a week from the peak level of >1,000 EU/l just after the start of sterilization.     

Ultrafiltration of dialysis fluid to obtain a sterile solution during hemodialysis

As the quality of water in the dialysis fluid varies considerably, and in view of the fact that endotoxin or active derivates can cause acute and chronic side effects in patients under chronic hemodialysis treatment, the dialysis fluid must be sterile and endotoxin free. The predialyzer fluid in 20 hemodialysis patients was investigated. The bacterial load was between 5/ml and 12,000/ml, the endotoxin concentration was high and extremely variable. Therefore, we introduced the ultrafiltration of the dialysis fluid by a polyamide hollow fiber membrane before entering the dialyzer. All samples were free of bacteria, and the concentration of endotoxin was lower than the detectable limit. With this procedure, we can obtain sterile dialysis fluid, which is endotoxin free. Our preliminary results showed that interleukin-1 in the patients was significantly (p less than 0.005) lower under ultrafiltration of the dialysis fluid than without ultrafiltration.     

High-permeable membranes and hypersensitivity-like reactions: role of dialysis fluid contamination

We have recently observed repeated hypersensitivity-like reactions (skin flush, face and tongue tingling, hypotension, and dispnea) during the first 5 min of dialysis in a small number of our dialysis population treated with high-flux membranes and traditional acetate dialysate. This prompted us to investigate the relationship between these reactions and the presence of contamination of the dialysate fluid. We hypothesized that in the presence of contaminated dialysate fluid and high-flux membranes backfiltration of pyrogens may occur through the membrane into the blood compartment, leading to hypersensitivity-like reactions. These events are more likely to occur at the onset of dialysis due to rapid changes of hydrostatic pressure gradients across the dialysis membranes. 6 out of 48 dialysis patients who experienced hypersensitivity-like reactions were followed for 4 weeks. During the 1st week they were treated with high-permeable membranes and during the 2nd week with cuprophane membranes. The dialysate showed high levels of contamination with bacteria and endotoxin during dialysis with both types of membranes (microbial count 4,123 +/- 2,756 and 1,991 +/- 1,950 colony-forming units/ml; endotoxin 26.2 +/- 8.4 and 23 +/- 4.2 endotoxin units/ml, respectively); however the symptoms occurred only during dialysis with high-flux membranes. This suggests that backfiltration of contaminated dialysate into the blood might have occurred during the early phases of dialysis only when using high-flux membranes, but not when using cuprophane membranes. To test this possibility we introduced a new dialyzer-rinsing device consisting of two simple connection lines which allow to rinse, in a concurrent manner, the dialysate and the blood compartments of the dialyzer with sterile saline solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of backfiltration rates during hemodialysis with highly permeable membranes.

A method for determining local transmembrane fluid movement in a commercial hemodialyzer at low dialysate flow rates by measuring changes along the dialyzer length in the local concentration of a marker macromolecule added to the dialysis solution has been developed. The method was evaluated in vitro at zero net ultrafiltration using dialyzers containing polysulfone (n = 4) and cuprophane (n = 3) membranes. The local concentration of the marker macromolecule along the dialyzer length was higher than the input dialysate concentration only during experiments with dialyzers containing polysulfone membranes. These observations provide direct empirical evidence that fluid movement in the dialysate to blood direction, i.e., backfiltration, occurs during hemodialysis with this highly permeable membrane. Net rates of backfiltration for the dialyzer containing polysulfone membrane were also calculated from changes in the local concentration of the marker macromolecule and mass balance considerations. The calculated backfiltration rates increased with increasing blood flow rate and trended upward with increasing dialysate flow rate. The described methodology provides a novel approach for the further characterization of fluid and solute transport during hemodialysis with highly permeable membranes.     

When good water goes bad: how it happens, clinical consequences and possible solutions

Dialysis fluid produced by state-of-the-art water preparation and distribution is contaminated with gram-negative bacteria and cytokine-inducing substances (CIS) derived from these microorganisms. The presence of a biofilm increases the risk of continuous contamination of dialysis fluid. Depending on the type of dialyzer membrane (cellulosic vs. synthetic) and the mode of dialysis (low flux vs. high flux with backfiltration), CIS may penetrate intact dialyzer membranes, induce cytokine production in the patient's blood and contribute to chronic inflammation associated with long-term hemodialysis therapy. Measures to improve the microbiological quality of dialysis fluid are: (1) the awareness of the problem and regular testing of dialysate samples using adequate methods; (2) disinfection of the entire water preparation and distribution system on a regular basis, replacement of biofilm-containing tubings, and (3) installation of ultrafilters in the dialysate circuit in particular when high-flux hemodialysis modalities are performed.     

Use of ultrapure dialysate in reduction of chronic inflammation during hemodialysis

Chronic inflammation contributes to the pathogenesis of several complications of hemodialysis therapy. It is thought that backfiltration of bacteria-derived contaminations during dialysis may induce a chronic inflammatory state. High-sensitivity C-reactive protein (hs-CRP) is one of the tools which can take a hold on such a chronic inflammatory condition. We examined the effect of ultrapure dialysate which contributes to chronic inflammation with hs-CRP and tried to reduce endotoxin (ET) levels at the end of the dialysate from 70 EU/l to <1.0 EU/l (ultrapure dialysate). Other dialysis conditions, except ET level, were fixed. We investigated the hs-CRP of 23 patients receiving regular dialysis before the use of ultrapure dialysate and 1 year after use of it prospectively. The data showed a significant decrease in the median value of the hs-CRP from 0.16 to 0.07 mg/dl (p < 0.05). The value of serum beta(2)-microglobulin decreased from 33.2 to 28.4 mg/dl (p < 0.01) and the hemoglobin level increased from 10.0 to 11.0 g/dl (p < 0.05). These results indicate that even a dialysate containing 70 EU/l of ET level may induce a chronic inflammatory state. hs-CRP is a very useful marker of chronic inflammation and the use of ultrapure dialysate is necessary to improve a chronic inflammatory state. The targeted ET level at the end of the dialysate should be set at < or = 1.0 EU/l.     

Control of secondary hyperparathyroidism during long-term hemodialysis

The hyperparathyroidism which occurs during long-term hemodialysis of patients with chronic renal failure has been successfully controlled without the need for subtotal parathyroidectomy. Utilizing a highly sensitive radioimmunoassay for plasma parathyroid hormone (iPTH), it was found that iPTH was correlated negatively with dialysate calcium concentration and positively with plasma phosphate concentration. Manipulation of these factors separately demonstrated that iPTH could be significantly decreased in two to three weeks by simultaneously decreasing plasma phosphate and increasing dialysate calcium. On the basis of calcium flux studies across the dialyzer, it was determined that the optimal calcium concentration in the dialysate was 8 mg/100 ml. Ten patients were treated by initially decreasing their plasma phosphate to less than 6 mg/100 ml (by giving them oral aluminum hydroxide) and using dialysis against a calcium concentration of 8 mg/100 ml. All patients have manifested a substantial and progressive decrease in plasma iPTH toward normal, with alleviation of bone pain and absence of complications, during use of this regimen.     

Dialysis hypoxemia. Role of dialyzer membrane and dialysate delivery system

Arterial blood gas values, carbon monoxide diffusion capacity, oxygen consumption, carbon dioxide production, respiratory quotient, minute ventilation, and pulmonary capillary blood flow were determined before and during hemodialysis. In addition, the effect of single passage through the dialyzer on blood carbon dioxide tension, pH, and bicarbonate concentration was evaluated. Acetate-based dialysate was used in all experiments. Cellulosic dialyzer with single-pass dialysate delivery system was used in one group, and polyacrylonitrile dialyzers with recirculating delivery system in another. Although hypoxemia occurred in both groups, it was more severe in the former group. Dialyzer carbon dioxide loss was significantly greater with single-pass dialysate delivery system and cellulosic dialyzers than with recirculating delivery system and polyacrylonitrile dialyzer. To differentiate the role of dialysate delivery system from that of the membrane, the experiments were repeated using recirculating delivery system and cellulosic dialyzer. This resulted in marked attenuation of hypoxemia and dialyzer carbon dioxide tension losses. Since other experimental conditions were the same, the observed differences were thought to be due to the difference in the mode of dialysate delivery. It thus appears that the mode of dialysate delivery per se can modify the changes in arterial oxygen tension during hemodialysis and should be added to the list of factors implicated in the genesis of dialysis hypoxemia.     

Hemodialysis-associated induction of cytokines

The interleukin hypothesis relates chronic pathology in long-term end-stage renal disease (ESRD) patients to repeated stimulation of mononuclear cell cytokine production during hemodialysis. In vitro experiments demonstrated different possible mechanisms involved in hemodialysis-associated cytokine induction: adherence of mononuclear cells to the dialyzer membrane; complement activation, and the passage of cytokine-inducing bacterial fragments from contaminated dialysate through the dialyzer membrane into the blood. Studies investigating mononuclear cells from ESRD patients ex vivo suggest that these cells become activated during hemodialysis with complement-activating membranes and that the type of dialyzer membrane may influence mononuclear cell cytokine production in response to endotoxin. According to biological assays, plasma levels of interleukin-1 but not interleukin-6 activity seem to be elevated in ESRD patients compared to normal subjects and may increase further during treatment depending on the choice of the dialyzer membrane. However, to date, partly due to insufficient assay sensitivity and circulating inhibitors, measurements of interleukin-1, interleukin-6 and tumor necrosis factor in plasma by specific immunoassays could not finally prove elevated plasma cytokine levels in ESRD patients. Since circulating mononuclear cells are a major source of cytokines, studying the activation of these cells ex vivo seems to be the best approach to study hemodialysis-associated cytokine induction.     

Endotoxin levels in cirrhotic rats with sterile and infected ascites.

Endotoxin levels were measured in sterile and bacterially infected ascites in a rat model of phenobarbital and carbon tetrachloride induced cirrhosis was used. An improved chromogenic substrate assay was used to measure endotoxin. All rat ascites specimens were positive for endotoxin. In culture-negative ascites (n = 8), it ranged from 0.05 EU/ml to 0.14 EU/ml (0.08 +/- 0.04 EU/ml, mean +/- SD) (Escherichia coli 0111:B4 endotoxin was used as a reference). In culture-positive ascites (premortem n = 3, postmortem n = 1), it ranged from 0.78 EU/ml to 1.8 EU/ml (1.29 +/- 0.59 EU/ml, mean +/- SD). All rats with premortem culture-positive ascites died within two days. This model is useful to study ascites endotoxin levels. In this study, increasing levels of ascites endotoxin correlated with spontaneous bacterial peritonitis and death.     

New trends in HDF therapies: validity of internal filtration-enhanced hemodialysis

Internal filtration-enhanced hemodialysis (IFEHD), defined as HD therapy using a dialyzer designed for enhanced internal filtration, seems to be more convenient in comparison with HDF therapies. In this paper, the validity of IFEHD was discussed during an analytical study and experimental studies. As a result, the maximum internal filtration flow rate (Q(IF)) value increased with a smaller inner diameter (D), a longer effective length (L(eff)), and a larger density ratio (DR) value of the hollow fibers. The validity of IFEHD was clarified by an analytical study and experimental studies. Development of a dialyzer with enhanced internal filtration, however, should take account of the patient's safety, and hemolysis and endotoxin invasion form the dialysate to the patient should be avoided.     

Changes in squamous cell carcinoma-related antigen levels before and after hemodialysis in relation to the model of dialyzer employed

OBJECTIVE: Although tumor marker levels in blood become elevated after hemodialysis as a result of the amount of fluid removed, serum squamous cell carcinoma-related antigen (SCC) levels have not been always reported to increase after hemodialysis. The purpose of this report is to determine whether there is a difference between the change of serum SCC levels before and after hemodialysis according to the model of dialyzer employed. PATIENTS AND METHODS: In ninety-four patients on hemodialysis (50 cases of diabetic nephropathy and 44 cases of glomerulonephritis), we examined serum SCC levels before and after hemodialysis. RESULTS: There was no overall difference between SCC levels before and after hemodialysis (3.2+/-1.5 ng/ml vs 3.3+/-1.7 ng/ml, p=0.2381). In patients treated with a cellulosic type membrane dialyzer (n=73), SCC levels after hemodialysis were higher than those before hemodialysis (3.7+/-1.7 ng/ml vs 3.5+/-1.5 ng/ml, p=0.0495). In patients treated with a synthetic type membrane (n=21), SCC levels after hemodialysis decreased when compared to those before hemodialysis (1.9+/-0.7 ng/ml vs 2.2+/-0.8 ng/ml, p=0.0018) and in all patients, the SCC levels after hemodialysis were lower than, or equal to, those before hemodialysis. CONCLUSION: The results suggest that the decline in serum SCC levels during hemodialysis treated with synthetic type membrane of dialyzers, concealed the increased SCC levels in hemoconcentration.     

Busulfan clearance in renal failure and hemodialysis

The impact of hemodialysis on the clearance of busulfan was determined in a patient with chronic renal failure undergoing autologous peripheral stem cell transplantation for non-Hodgkin's lymphoma. The extraction ratio for busulfan across the dialyzer was 0.530 +/- 0.026 at a blood flow of 400 ml/min, which corresponds to a hemodialysis clearance of 2.23 +/- 0.11 ml/min/kg body weight. Apparent oral clearance of busulfan without hemodialysis was 3.38 +/- 0.56 ml/min/kg. Thus, a 4 h hemodialysis session enhanced the apparent oral clearance of busulfan by 65%. We conclude that hemodialysis effectively removes busulfan from circulating blood, but a standard hemodialysis period (ie, 4 h) does not significantly alter busulfan exposure. Bone Marrow Transplantation(2000) 25, 201-203.     

Pathophysiologic quantities of endotoxin-induced tumor necrosis factor-alpha release in whole blood from patients with chronic heart failure

Bacterial endotoxin activity is elevated in patients with decompensated chronic heart failure (HF) and acts as a potent stimulus for immune activation. We sought to determine whether endotoxin, at an activity level seen in vivo (around 0.6 EU/ml), is sufficient to stimulate the secretion of tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha soluble receptor (sTNFR2) in ex vivo whole blood from patients with HF. We studied 15 patients with HF (aged 65 +/- 1.9 years, New York Heart Association class 2.1 +/- 0.3, left ventricular ejection fraction 31 +/- 5%; mean +/- SEM), of whom 5 had cardiac cachexia, and 7 healthy control subjects (59 +/- 5 years, p = NS). Reference endotoxin was added to venous blood at concentrations of 0.6, 1.0, and 3.0 EU/ml, and was incubated for 6 hours. Endotoxin induced a dose-dependent increase in TNF-alpha release (p <0.05 in all groups). Patients with noncachectic HF produced significantly more TNF-alpha compared with controls after stimulation with 0.6, 1.0, and 3.0 EU/ml of endotoxin (113 +/- 46 vs 22 +/- 4 [p = 0.009], 149 +/- 48 vs 34 +/- 4 [p = 0.002], and 328 +/- 88 vs 89 +/- 16 pg/ml [p = 0.002], respectively; mean +/- SEM). Patients with cardiac cachexia produced significantly less TNF-alpha compared with patients without cardiac cachexia for all given concentrations (all p <0.05, analysis of variance p = 0.02). Production of sTNFR2 was greater at all concentrations of endotoxin versus controls (all p <0.05, analysis of variance p = 0.002). Plasma endotoxin levels were higher in patients with cardiac cachexia (4.3 times higher than in control subjects, p <0.005). Thus, low endotoxin activity, at levels seen in vivo in patients with HF, induces significant TNF-alpha and sTNFR2 production ex vivo. These results suggest that elevated plasma endotoxin activity observed in patients with HF is of pathophysiologic relevance.     

Implementing On-Line Hemodiafiltration as a Renal Replacement Therapy for ICU Acute Renal Failure: A Single-Center Report of Feasibility, Safety and Hemodynamic Tolerance over a Seven-Year Period

Background/Aims: On-line hemodiafiltration (HDF) is not yet routinely used in ICUs given the potential risk of microbial contamination of dialysis fluids. We evaluated the safety and the tolerance of its use in our ICU. Methods: A weekly measurement of bacterial growth (CFU/ml) and endotoxin level (endotoxin units/ml) was performed in dialysis fluids over a 7-year period. Intradialytic hypotensive events and pyrogenic reactions were collected during 466 on-line HDF sessions. Results: A bacterial count <0.1 CFU/ml was achieved in 977/978, 288/290, and 278/280, and an endotoxin level <0.03 endotoxin units/ml in 564/576, 330/337 and 318/323 ultrapure water, dialysate, and infusate samples, respectively. Seventy-six intradialytic hypotensive events but no pyrogenic reaction occurred. Conclusion: The great majority of dialysis fluid samples were considered suitable with a 99% compliance rate. Use of on-line HDF, at a large scale of dialysate and infusate flows, is well tolerated and may be safely performed in critically ill.     

Effects of Dialysis Purity on Uremic Dyslipidemia

Dyslipidemia, a prominent feature of end-stage renal disease, is considered a risk factor for premature atherosclerosis in hemodialysis (HD) patients. Dyslipidemia is related to loss of kidney function as well as use of low-flux cellulosic dialyzer membranes, but the effects of dialysate purity are unknown. Forty-eight incident HD patients started high-flux polysulfone maintenance HD, either with conventional (potentially contaminated) or with on-line produced ultrapure dialysate. The quality of the dialysis fluid (CFU/mL, endotoxin concentration), markers of inflammation (C-reactive protein, Il-6), and parameters of the lipid profile and oxidative stress (oxidized low-density lipoprotein) were measured before initiation of HD, and after 6, 12 and 24 months on HD. Compared to baseline, treatment with conventional (mildly contaminated) dialysate significantly increased the uremic low-grade systemic inflammatory response syndrome (SIRS), augmented uremic dyslipidemia (triglycerides by +21%, and high-density lipoprotein (HDL) cholesterol by −10%) and enhanced oxidative stress. In contrast, the use of ultrapure dialysate significantly decreased uremia-associated SIRS, dyslipidemia (triglycerides −7% and HDL cholesterol +11%) and oxidative stress. Ultrapure dialysis fluid improves potential parameters of cardiovascular risk by decreasing inflammatory reactions, improving uremic dyslipidemia and lowering oxidative stress.