Occurrence of endotoxin in dialysis fluid from 39 dialysis units

Journal

of Hospital

Infection

Occurrence

Lena

of endotoxin dialysis

Kulander*,

Departments

of

(1993) 24, 29-37

U. Nisbethf, *Clinical

in dialysis units

fluid

B. G. Danielssont

Chemistry and fInterna1 Hospital, Uppsala, Sweden

Accepted for publication

15 February

from

39

and d. Eriksson* Medicine,

Uniaersity

1993

Summary: Endotoxin exposure during haemodialysis may cause acute and chronic adverse reactions. In order to estimate the risk to the patient, samples of dialvsis fluid from 39 of the 45 dialvsis units in Sweden were analvsed bv the chromogenic Limulus amoebocyte lysate assay. Higher levels were obtained after the usual weekend shutdowns. The length of the tubing delivering the reverse osmosis water seemed to influence the extent of contamination. Fifty-nine percent of the units showed low mean endotoxin levels (i.e. mean concentration below the recommended limit in Sweden: ~25 ng ll’), while 18% of units had high levels (mean concentration > 100 ng 1-l). Keywords:

Dialysis

fluid;

endotoxin;

haemodialysis;

limulus

assay.

Introduction Endotoxins are lipopolysaccharides derived from the outer cell wall of degraded Gram-negative bacteria. They have profound biological effects with ability to activate different cascade systems such as coagulation and complement. The toxicity of the lipopolysaccharide molecule is caused by lipid A, the region that is least variable in structure.“2 Thus, there is no variability in the endotoxin toxicity between different known Gram-negative bacteria. Depending on the degree of endotoxaemia, several clinical symptoms may develop, such as fever, chills, headache, myalgia, hypotension, metabolic acidosis, disseminated intravascular coagulation and septic shock. A patient on regular haemodialysis treatment (RDT) is exposed to approximately 400 1 of dialysis fluid each week through the extracorporeal Bicarbonate-containing dialysis fluid (BD) and high-flux circulation. membranes, now used more widely, have several advantages. However, the bicarbonate favours bacterial growth and the larger pore size of the high-flux membrane probably enhances the risk of pyrogenic reactions. Correspondence to: Dr Lena 85 Uppsala, Sweden

Kulander,

Department

of Clinical

Chemistry,

University

Hospital,

S-751

30

L. Kulander

et al.

Consequently the need for ultrapure atoxic water, and sterile nonpyrogenic dialysis fluid has increased. 3,4To date it has not been possible to establish a safe limit for pyrogenic contaminants in dialysis fluid, i.e. which level of endotoxin in dialysis fluid passing the dialyser will elicit adverse reactions acutely or chronically, although a few countries, including Sweden, have introduced a limit (< 25 ng 1-l) equivalent to that for intravenous preparations. Gram-negative bacteria dominate in cultures from dialysis water and liberate endotoxin. Among the Gram-negative bacteria Pseudomonas species are most common, i.e. Pseudomonas jkorescens, Pseudomonas maltophilia, Pseudomonas putida, Pseudomonas stutzeri and Pseudomonas vesicularis. Other commonly isolated bacteria are Moraxella and Alcaligenes spp.‘v6 It is still not clear to what extent endotoxin, Limulus-reactive material (LAL-RM), penetrates or acts through different modern dialysers. Under normal conditions endotoxin exists as large aggregates (100-l 000 kD). However, smaller and more bioactive fragments occur (l-10 kD). In vitro, acid hydrolysis is required to liberate the smallest active part, lipid A (2-3 kD). The addition of surfactants and the removal of calcium and magnesium ions result in disaggregation of the endotoxin complex.7 In-vitro as well as in-vivo studies8v9 have shown that endotoxincontaminated dialysis fluid activates monocytes in the bloodstream, thereby eliciting the release of cytokines, e.g. interleukin-1 (IL-l) and tumour necrosis factor (TNF). IL-l and TNF are able to induce the production of interleukin-6 (I L-6) by human monocytes. lo Increased IL-6 levels in sera of haemodialysed patients have been described.” These cytokines mediate fever and the acute phase reaction. Other factors such as acetate-buffered dialysis fluid, complement and the physical nature of the membrane have also been found to activate monocytes. ‘* Well-known factors favouring bacterial growth in the water system are carbon filters, dead spaces and other parts with low circulation, e.g. tanks and taps. Air traps and water heaters in certain dialysis machines are difficult to disinfect adequately. Biofilms consisting of an amorphous material containing bacteria occur in tubing and tanks; these films are extremely difficult to eradicate chemically or physically.13 In this study we aimed to determine the occurrence and extent of endotoxin contamination in dialysis fluid among the majority of dialysis units in Sweden and estimate the risk to the patient. Especially, we wished to evaluate the influence of tubing length and observe any difference between Mondays and Fridays, as a function of weekend shutdowns. Methods

Equipment and haemodialysis procedures Reverse osmosis water was usually prepared and thereafter in a separate part of each dialysis unit. The most common

stored in a tank RO-systems in

Endotoxin

Sweden are Gambro, water is delivered by finally prepared, i.e. The most commonly

in dialysis

fluid

31

Culligan, Millipore and Vattenteknik. From the tank, tubing to each dialysis machine in which the fluid is heated to 37°C and electrolyte and buffer-balanced. used dialysis machines are Gambro and Fresenius.

Sampling protocol Samples of dialysis fluid from the majority (39 of 45) of the dialysis units in Sweden, were collected during two consecutive weeks. The samples were collected before entering the dialyser prior to the start of haemodialysis. Double samples were collected on Mondays and Fridays, both close to and as far as possible from the water source. From each unit 2 x 8 samples were collected on each occasion and a total of 640 samples was thus collected in total. From one of the units we received another 16 samples. The dialysis fluid was collected in evacuated glass tubes (Becton-Dickinson) and immediately stored at - 20°C. Before the samples were collected, the tubes were shown to be endotoxin-free (contained < 5 ng 1-l endotoxin). Laboratory methods For quantification of endotoxin in dialysis fluid, Limulus amoebocyte lysate (LAL) and a chromogenic substrate were employed, according to a technique described by Friberger,14 which we have modified. Limulus lysate from M. A. Bioproducts, Walkersville, Maryland, USA and a chromogenic substrate were supplied by Kabi Diagnostica AB, Mijlndal, Sweden. Escherichia coli 0 11 l:B4 was used as a standard, and the activity of 1 ng was equivalent to 12 endotoxin units (EU) compared with Food and Drug Administration standard EC-S/USP lot F. All glassware was covered with aluminium foil and heated in a hot-air oven at + 200°C for > 4 h. From the stock solution of the endotoxin standard, serial dilutions were made for the standard curve. The concentration covered the range of O-100 ng 1-l. The samples and the standards were diluted 1: 10 with sterile endotoxin-free water to eliminate any inhibition of the assay. LAL was reconstituted in 1.4 ml of sterile endotoxin-free water; 100 ~1 lysate was incubated with a 100-~1 sample or standard in a block heater at 37°C (step 1). After 20 min 200 ~1 substrate-buffer was added (consisting of one volume of chromogenic substrate (2.0 mmol 1-i) mixed with one volume of TRIS buffer (50mmol l-‘, pH 9.0) (step 2). Five min later the reaction was stopped by adding 200 ~1 of 20% acetic acid. The yellow colour was read at 405 nm. There was a linearity of extinction between 5 and 100 ng ll’. The intra-assay coefficient of variation, the concentration level 10 ng I-‘, was 5.1%, and the corresponding interassay variation 10.7%. Statistical analysis Significance of differences between endotoxin concentrations was evaluated by Student’s t-test for paired observations (Table I) and Wilcoxon signed ranks test (Table III and Table IV).

L. Kulander

32

et al.

Table I. Endotoxin concentrations determined by the use of undiluted and diluted (1:lO) samples of dialysis fluid. Endotoxin Sample

no.

1 2 3 4 5 6

Undiluted

concentration sample

Diluted

SEM

sample

5 (:

92 146

:

;ri 208 57

4

Mean f

(ng 1-l)

2.2f0.8

106*24.5

Dilution was performed with endotoxin-free tion. Student’s t-test on paired observations

water for injec(I’< 0.01).

Results

Six undiluted samples of dialysis fluid gave a mean endotoxin concentration ( f SEM) of 2.2 f 0.8 ng 1-l (Table I). The endotoxin concentration when the samples were diluted 1 :lO with endotoxin-free water was 106-O& 24.5 ng 1-l. The difference was statistically significant (PC 0.01). Thus, the dialysis fluid gave rise to an inhibition of the limulus assay. Further dilution (1:20 and 1:SO) did not result in higher levels. Because of the inhibition all samples of dialysis fluid were diluted 1: 10 with endotoxin-free water before the assay. Sixteen of the 39 dialysis units had mean endotoxin concentrations of >25ng 1-l in the dialysis fluid (Table II). The highest mean level of endotoxin found in dialysis fluid from one unit was 224ng 1-l (range 3-l 160 ng 1-l). In seven high scoring units the dialysis fluid contained > 100 ng 1-l endotoxin. In three of these dialysis units, the mean value (105, 139 and 178 ng 1-l endotoxin, respectively) was biased by a single high scoring day with high concentrations, 60&l 560 ng 1-l. However, the mean Table

II.

Distribution

Mean endotoxin concentration (ng 1-l) in samples from a unit o-25 26-50 51-100 101-150 151-200 201-250

of endotoxin

concentration Sweden

in dialysis

fluid

from

39 dialysis

units in

Number of dialysis units in each category

Total number of samples from these units

Mean ( f SEM) endotoxin concentration (ng I-‘) in these samples

Range of endotoxin concentrations (ng 1-l) in these samples

23 6 3 4 2 1

377 92

8.8 zt 0.7 32.0% 2.4 57.2f9.4 129.4f20.3 175.0f 56.3 223.6 f 89.2

O-126 O-132 l-330 Ck765 14-1560 3-1160

2: 31 16

Endotoxin

in dialysis

fluid

33

Table III. fluid taken

Endotoxin concentration in samples of dialysis close to and distant from the water production unit.

Endotoxin bs 1-7

concentration

Median value Mean 4~SEM Range Number of samples

Close to unit 6.8 25.8 * 3.7 C7.55 320

Distant from unit 16.3 61.7f9.6 O-1560 320

The difference between endotoxin concentrations is significant when tested with Wilcoxon signed ranks test (PC 0.0002).

values would still be high (27, 61 and 79 ng I-’ endotoxin, respectively), if the highest value were discarded. All these samples were taken distant from the water source. In two cases the highly contaminated samples were taken on Mondays and in one case on a Friday. Samples of dialysis fluid taken distant from the water source contained significantly higher (P < 0.0002) endotoxin levels than samples taken close to the water source (Table III). The 95% confidence interval for the mean of the distant samples was 34.8-88.6 ng 1-l and for the nearer samples it was 154-36.1 ng 1-l. Samples of dialysis fluid collected on Mondays contained significantly higher (P~O.005) endotoxin concentration than samples taken on Fridays interval for the mean of the Monday (Table IV). The 950/ o confidence determinations was 29.6-83.3 ng 1-l and for the Friday determinations it was 20.1-42.0 ng I-‘. Discussion

The present investigation of dialysis fluid from the majority (39 of 45) of the dialysis units in Sweden showed that 16 of the units had mean endotoxin concentrations of > 25 ng 1-l in the dialysis fluid. Of these, seven had levels of > 100 ng 1-l. In one dialysis unit the mean endotoxin concentration was Table

IV.

Endotoxin (ng 1-7

Endotoxin concentration in samples of dialysis fluid taken on Mondays and on Fridays concentration

Median value Mean f SEM Range No. of samples

Mondays

Fridays

12.8 56.5*9.6 Cl560 320

9.3 31.0f3.9 O-683 320

The difference between endotoxin concentrations is significant when tested with Wilcoxon signed ranks test (PC 0~00.5).

34

L. Kulander

et al.

224 ng 1-l. The study also included a comparison between the dialysis fluids on Mondays and Fridays, and between samples taken close to and distant from the water source. The endotoxin levels were found to be significantly higher on Mondays than on Fridays (Table IV) and in samples taken furthest from the water source compared with those taken close to the source (Table III). The probable reason for the former was that the water circuit lay unused over the weekend, an interval that would permit bacterial growth. Not surprisingly, long tubing seems to promote growth of bacteria, as expressed by higher endotoxin-levels. This is in accordance with the observations of a biofilm containing bacteria in tubing.‘3”5 In a single haemodialysis treatment about 120 1 of dialysis fluid is used. If the dialysis fluid holds an endotoxin concentration of 224 ng l-‘, as found in one dialysis unit, a total of 27 ltg of endotoxin will be transferred to the dialysis filter. If one assumes that 1% of this passes through the dialysis filter and reaches the patient’s circulation, the ‘endotoxin dose’ will be about 3.8 ng kg-’ body weight (b.w.) for a 70 kg person. This is well above the lowest reported endotoxin concentration (O*l-0.5 ng kg-’ b.w.) that elicited fever.16 It has been shown that between O-1 and 1.9% of the endotoxin in contaminated dialysis fluid passes through AN 69, polysulfone and cuprophan dialysis membranes, in an in-vitro dialysis circuit.3,‘7 In another the passage of endotoxin during study, Sawada et al.” have investigated continuous flow through a microporous polyethylene hollow-fibre (EHF) membrane. When 5 1 of endotoxin-contaminated tap water had passed through the membrane, endotoxin was observed in the filtrate, and during continuous passage an exponential increase of endotoxin was observed. On the other hand, Bommer et aZ.19were not able to demonstrate the passage of LAL-RM. Endotoxin was probably excluded because of size and charge and adsorbed to the outer membrane layer.20 These studies are contradictory. One possible explanation is that endotoxin molecules are adsorbed to the ‘binding sites’ on the membrane, and then when all binding sites are occupied the excess endotoxin can pass through the filter. It is possible that these workers 19s2’did not exceed the binding capacity of the membranes, in contrast to the other authors. Nisbeth et aL21 and Watzke et a1.22measured the endotoxin concentration in the blood in patients undergoing haemodialysis and found that the concentration increased after this treatment. In-vitro studies by Dinarello12 and by Lonnemann et aZ.* showed that when circulated through the blood side of a closed-loop dialysis circuit, fresh human blood containing monocytes was stimulated to produce interleukin-1 (IL-l) when endotoxin was present in the dialysis fluid compartment. The IL-l production started when the endotoxin concentration was as low as 50 ng 1-l and was already observed after 15 min of circulation. The activated monocytes also produce beta-2-microglobulin which accumulates in RDT patients and may result in complications from the associated amyloidosis.23!24 The question is what endotoxin level in the dialysis fluid is tolerated by the patient. Perhaps even

Endotoxin

in dialysis

fluid

35

a low-grade recurrent exposure is a hazard to health. The immune system is generally suppressed in uraemia. 25,26 Despite the reduced monocyte response to endotoxin, patients on RDT exhibit elevated predialysis levels of IL-l*’ and circulating endotoxin. 21,22 Further evidence for endotoxin exposure is the fact that RDT patients have elevated antibody levels against endotoxin.** The incidence of fever during haemodialysis is also indirect evidence. With the use of ultrapure bicarbonate dialysis fluid (BD) during high-flux dialysis a lower incidence of fever ( < 0.3%) was found, in comparison with 1.4% with standard BD containing endotoxin (So-250 ng 1-‘).4 The incidence of fever during conventional haemodialysis was 4.8%, but only 0.8% for patients receiving haemofiltration.29 The chromogenic Limulus assay seems to be a sensitive and specific method for endotoxin testing of dialysis fluids. Endotoxin analysis of 2 x 8 samples of dialysis fluid from dialysis sessions in each of 39 units (320 double samples) showed low mean values (< 25 ng 1-l) for 59% of the units. About 8% had very high mean endotoxin levels (150-250 ng 1-l) and these fluids may cause acute pyrogenic reactions even if only 1% of the total endotoxin would pass the dialyser membrane (calculated on a patient of 70 kg b.w. exposed to 120 1 dialysis fluid). In the present study, we found that endotoxin contamination was influenced by factors in the water production system such as water stagnation and design of the distribution system. Of course, bacterial growth in dialysis fluid above the recommended level should not be accepted. The first remedial action is usually to perform an extra chemical cleaning of the dialysis machine, and of the water system to the reverse osmosis filter. The tubing is disinfected by heating to at least 90°C. Further bacteriological sampling should then be carried out. However, older dialysis machines may be difficult to clean satisfactorily and may have to be replaced if there is considerable bacterial growth despite repeated disinfection. between bacterial count and Klein et ~1.~ did not find any correlation endotoxin levels in the dialysis fluid. This is probably due to the fact that the bacterial count only measures living bacteria, while endotoxins on the other hand are derived from both living and dead bacteria. In our opinion, endotoxin quantification of dialysis fluid is a valuable complement to bacterial count, especially as the Limulus method can be performed within 3-4 h, compared with 24-48 h for a culture-derived bacterial count. We thank Dr Torsten Aronsson for expert statistical advice. The present work was supported by grants from the Patients Association of Kidney Disease in CUWX-countries, Sweden. Reagents were provided by KABI Diagnostica AB, Molndal, Sweden.

References 1. Brade H, antigenicity

Brade L, Schade U, et al. Structure, endotoxicity, immunogenicity of bacterial lipopolysaccharides (endotoxins, O-antigens). In: Levin

and J, ten

L. Kulander

36 Cate

JW,

Biiller

pathophysiological

HR,

van

Deventer

SJH,

et al. Sturk

A.,

Eds.

effects, clinical significance, and pharmacological

Bacterial endotoxins: control. New York:

Alan R. Liss Inc. 1988; 17-45. 2. Rietschel E Th, Brade H. Bacterial endotoxins. An integral part of many bacteria, these molecules are at once brutal and beneficial to humans. Efforts are under way to block the bad effects and harness the good. Scienti$c American 1992; 267(2): 26-33. 3. Man NK, Cianconi C, Faivre JM, et al. Risks and hazards of contaminated dialysate associated with high flux membranes. In Buccianti G, Ed. Prevention in Nephrology. Milan: Masson Italia Editori 1987; 227-234. 4. Mion CM, Canaud B, Garred LJ, Stec F, Nguyen QV. Sterile and pyrogen free bicarbonate dialysate: a necessity for hemodialysis today. Adv Nephrol 1990; 19: 275-314. 5. Harding GB, Klein E, Pass T, Wright R, Million C. Endotoxin and bacterial contamination of dialysis center water and dialysate; a cross sectional survey. Int J

Artificial

Organs 1990; 13: 3943.

6. Klein E, Pass T, Harding GB, Wright R, Million C. Microbial and endotoxin Organs contamination in water and dialysate in the Central United States. Artificial 1990; 14: 85-94. 7. Pearson FC, Dubczak J, Weary M, Anderson J. Determination of endotoxin levels and their impact on interleukin-1 generation in continuous ambulatory peritoneal dialysis Blood Purij 1988; 6: 207-212. and hemodialysis. 8. Lonneman G, Binge1 M, Floege J, Koch KM, Shaldon S, Dinarello CA. Detection of endotoxin-like interleukin-l-inducing activity during in vitro dialysis. Kidney Int 1988; 33: 29-35. 9. Binge1 M, Lonneman G, Koch KM, Dinarello CA, Shaldon S. Plasma interleukin-1 activity during hemodialysis: the influence of dialysis membranes. Nephron 1988; 50: 273-276. 10. Shalaby MR, Waage A, Aarden L, Espevik T. Endotoxin, tumor necrosis factor-u and interleukin-1 induce interleukin-6 production in vivo. Clin Immun Immunopathol 1988; 53: 488498. 11. Cavaillon JM, Poignet JL, Fitting C, Delons S. Serum interleukin-6 in long-term hemodialyzed patients. Nephron 1992; 60: 307-3 13. 12. Dinarello CA. Interleukin-l-its multiple biological effects and its association with hemodialysis. Blood Purij 1988; 6: 164-172. 13. Du Molin GC, Coleman EC, Hedley-Whyte J. Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene. Appl Env Microbial 1987; 53: 1322-1326. 14. Friberger P. The design of a reliable endotoxin test. In: ten Cate JW, Biiller HR, Sturk A, Levin J, Eds, Bacterial endotoxins: structure, biomedical significance and detection with the Limulus amebocyte lysate test. New York: Alan R. Liss Inc. 1985; 139-149. 15. Phillips G, Hudson S, Stewart WK. Microbial growth and blockage of sub-floor drains in a renal dialysis centre: a problem highlighted. J Hasp Inject 1992; 21(3): 193-198. 16. Elin RJ, Wolff SM, McAdam KPWJ, Chedid L, Audibert F, Bernard C, Oberling F. Properties of reference Escherichia coli endotoxin and its phthalylated derivative in humans. TJ Inject Dis 1981; 144: 329-336. 17. Laude-Sharp M, Caroff M, Simard L, Pusineri C, Kazatchkine M, Haeffner-Cavaillon N. Induction of IL-l during hemodialysis: Transmembrane passage of intact endotoxins (LPS). Kidney Int 1990; 38: 1089-1094. 18. Sawada Y, Fujii R, Igami I, Kawai A, Kamiki T, Niwa M. Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane. Appl Env Microbial 1986; 51: 813-820. 19. Bommer J, Becker KP, Urbaschek R, Ritz E, Urbaschek B. No evidence for endotoxin transfer across high flux polysulfone membranes. Clin Nephrol 1987; 27: 278-282. 20. Schindler R, Dinarello CA. A method for removing interleukin-1 and tumor necrosis factor-inducing substances from bacterial cultures by ultrafiltration with polysulfone. J Immunol Meth 1989; 116: 159-165. 21. Nisbeth U, Hillgren R, Eriksson 0, Danielsson BG. Endotoxemia in chronic renal failure. Nephron 1987; 45: 93-97. 22. Watzke H, Mayer G, Schwarz HP, et al. Bacterial contamination of dialysate in dialysis-associated endotoxaemia. J Hosp Znject 1989; 13: 109-l 15.

Endotoxin

in dialysis

fluid

37

23. Knudsen PJ, Ah-Kau N, Zhuoru L. Beta-2-microglobulin synthesis is increased during activation of human monocytes. Blood Purif 1988; 6: 178-187. 24. Stein G, Schneider A, Thoss K, et al. Beta-2-microglobulin-derived amyloidosis: onset, distribution and clinical features in 13 hemodialysed patients. Nephron 1992; 60: 274-280. 2.5. Dobbelstein H. Immune system in uremia. Nephron 1976; 17: 4099414. 26. Kunori T, Fehrman I, Ringden 0, Moller E. In vitro characterization of immunological responsiveness of uremic patients Nephron 1980; 26: 236239. 27. Blumenstein M, Schmidt B, Ward RA, Ziegler-Heitbrock HWL, Gurland HJ. Altered interleukin-1 production in patients undergoing hemodialysis. L\Tephron 1988; 50: 2777281. 28. Yamagami S, Adachi T, Sugimura T, et al. Detection of endotoxin antibody in long-term dialysis patients. Int J Artificial Organs 1990; 13: 205-210. 29. Schaefer K, von Herrath D, Huller M, Pauls A. The occurrence of fever during hemodialysis and hemofiltration. A comparative study. Int J Artijicinl Organs 1986; 9: 247-250.