- Email: [email protected]
Pseudomonas aeruginosa bacteremia in a dialysis unit
Pseudomonas aeruginosa Bacteremia in a Dialysis Unit II. Relationship
to Reuse of Coils
JON P. WAGNILD. M.D. PETER MCDONALD, M.D.’ WILLIAM A. CRAIG, M.D. CAROLINE JOHNSON, B.A. MICHAEL HANLEY, M.D.’ STEPHEN J. UMAN. M.D.* V. RAMGOPAL. M.B.B.S. GREGORY J. BEIRNE, M.D. Madison. Wisconsin
Blood for culture was obtained over a six week period from 17 patients undergoing long-term hemodialysts. Bacteremia was detected during 18 of 201 dialyses. Blood drawn during fifleen of these dialyses contained Pseudomonas aen@osa. Ten of the 17 pabents (59 per cent) had a Pseudomonas bacteremia some time during the study. Only one patient was symptomatic. The frequency of positlve cultures was related to reuse of co&. No cuttues were positive until after the fifth use, but by the tenth use, 41 per cent of the dialyses were associated wtth bacteremia. All coils that were used repeatedly and 32 of 48 of those used only once, grew Ps. aeruginosa when filled wlth media and Incubated. This suggests that the coils were inoculated during dialysis and that benzalkonium chloride, the sterilizing agent, was unable to eradicate this organism. With repeated uses, the number of residual bacteria in the coil became large enough to cause detectable bacteremia during dialysis. Dialysis coils are reused in many centers to decrease
From the Renal and Infectious Disease Units of the Medical Service, Veterans Administration Hospital: and the Department of Medicine, University of Wisconsin Schccl of Medicine, Medii, Wisconsin. Requests for reprints should be addressed to Dr. Jon P. Wagnild. Department of Medicine. Veterans Administration Hospital. 2500 Overlook Terrace, Madison, Wisconsin 53705. Manuscript accepted August 24, 1976. ’ Present address: Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000. ’ Present address: Department of Medicine, Nephrology Service, University of Texas Health Sciences Center, Dallas, Texas 75235. t Present address: 6360 Wilshire Boulevard, Los Angeles, California 90048.
672
May 1977
the high cost of hemodialysis. Several different methods of cleaning and storing coils have been proposed, and reuse of up to 15 times without significant complications or impairment in clearance characteristics has been reported [i-5]. In our dialysis center we have reused coils for over eight years without apparent ill effects and with considerable savings [5]. Coils were cleaned and stored using benzalkonium chloride as the sterilizing agent. However, over a recent six month period Pseudomonas bacteremia developed in four of 20 patients in w center, and one dled from Pseudomonas endocarditis [6]. To determine the source of the bacteremia, we undertook a bacteriologic survey of the hemodialysis unit. This included personnel, dialysate. machines, delivery system, coils, sterilization technic and, finally, the systematic collection of blood culties throughout each dialysis on all patients over a six week period. In this report we present the results of the survey involving cultures of the blood, coil and dialysate. In a companion paper we deal with the microbiologic studies of dialysate, machines and personnel [ 6 I. METHODS Seventeen patients with a stable condiiion undergoing lonpterm hemodiilysis were surveyed. All were dialyzed via a percutaneous puncture of a forearm arteriovenous fistula. For the first four weeks of the study, blood for culture was obtained from all 17 patients at the start of dialysis and at 2 hour intervals throughout each dialysis. Blood samples were drawn from the arterial line. Pediatric blood culture tubes (Vacutainer Culture Tube, Gentec Hospital Supply) were used which required only 2 ml of blood and thereby limited tie
The American Journal of Mediclne
Volume 62
PS
AERUGINOSA
total amount of blood withdrawn per 6 hour dialysis to 8 ml. Blood pressure and oral temperature measurements were recorded at each sampling time. Dialysate samples obtained from the coil compartment before dialysis were cultured on dip slides (Uricult. Medical Technology Corp.): dialysate samples obtained after dialysis were cultured on both dip slides and trypticase soy agar plates. The study was then continued an additional two weeks during which blood for culture was drawn only at the end of dialysis. This was from a group of patients whose coils were reused more than five times. If a blood culture became positive during this latter period, the dialysis unit was notified, and coils were either discarded or filled with media and cultured. A description of the dialysis unit has already been given [S]. Briefly, it is an eight station unit dosigned primarily for long-term hemodialysis. Approximately 19 patients undergo dialysis two to three times per week using Travenol RSPrm artificial kidney machines. Either a central proportioning system or bulk tanks are used for dialysate delivery. Cuprophane membrane coil dialyzers were routinely reused up to 10 times. Machines are interchanged between patients except in those suspected of or known to have infection or hepatitis. Coils to be reused were cleaned at the end of dialysis according to the following procedure. Blood remaining in the coil was flushed out with a saline solution made from tap water. When clear, the coil was filled with a solution of benzalkonitrn chloride in tap water (1:750), and stored at 4°C in a pail containing the same solution. Immediately prior to use, the benzalkonium chloride was poured off and the outside surface of the coils rinsed. They were then flushed with 3 liters of sterile distilled water and 1 liter of heparinized saline solution. During the infusion of a second liter of heparinized saline sofution, the coils were pressurt+cbecked and primed. Samples for culture were routinely obtained from the saline effluent from the coil prior to connecting the patient for dialysis. Culture Methods. Blood cultures were vented and incubated at 37% they were examined at 24 and 48 hours, and at one and two weeks. Dip slides of dialysis fluid were also incubated at 37’%, and the density was read at 24 hours. Samples of dialysate were refrigerated at 4°C and cultured within 24 hours. The predominating organisms were then picked and identified. Coils were cultured as follows: Initially, single and multiple use coils were cleaned and stored in the usual fashion, and prepared several days later for use. Trypticase soy broth (TSB) was then drawn into the coils from a farge flask utilizing the blood perfusion pumps, the blood tubings were clamped and the coils incubated at 37’C. Because of possible contamination during the cleaning or sterilizing procedure, a group of single use unsterilized coils was taken to the laboratory following dialysis, filled with TSB, clamped and incubated at 37’C for up to 10 days. Samples were subcultured at intervals by needle aspiration from the tubing. Media was drained from the coil at 10 days and further subcultured if earlier samples were negative for Ps. aeruginosa. All positive cultures were identified in the Clinical Microbiology Laboratory. Two to 6 colonies were individually subcultured from each blood and coil culture positive for Ps. aeruginosa. lmmunotyping was performed on each isolate
TABLE
average
BACTEREMIA
IN A DIALYSIS
Summary
I
UNIT
II
WAGNILD
ET AL
of Blood Cultures Positive for
Pseudomonas
with
Reuse of Coils
.._ Number of Times Coils were Used Data Patients studied DlalYSf?t studled P0SltlVe cultures Dialyses positive PntVmts positive
1
2
3
4
5
6
7
8
9
10
16
15
14
17
16
15
16
15
15
14
22
24
19
19
18
73
23
21
16
II
0
0
0
0
0
1
1
2
4
0
0
0
0
0
5
4
10
25
41
0
0
0
I)
0
7
6
13
27
50
(no.) ino. 7
(no.) 1%) (%I
by the slide agglutination method utilizing the antibody system described by Fischer et al. [7] using seven type-specific antiserums provided by t-f. B. Devlin, Ph.D., Parke-Davis Laboratories, Detroit, Mich. Resistance of Pseudomonas isolates to benzalkonium chloride was determined by the method of Adair et al. [ 81. Resistance is defined as growth in the presence of 1,000 pg/ml of benzalkoniumchloride.
RESULTS Blood Cultures. Blood for culture was obtained from 17 patients undergoing a total of 20 1 dialyses (Table I). During 170 of these dialyses, blood samples were drawn at the start and at 2 hour intervals thereafter; in the remaining 31 dialyses, only two blood samples for cultures were obtained at the end of dialysis. When examined at 24 and 48 hours, almost all blood cultures were negative. Only after incubation for longer periods of time, generally three to seven days, was growth sufficient to be detected. By this time the coils had been reused or discarded. Positive blood cultures were obtained in 18 of the 201 dialyses (9 per cent). Ps. aeruginosa was present in 15, and diphtheroids, Staphylococcus epidermidis and Klebsiella species were each isolated once. The diphtheroids and Staphylococcus epidermidis were presumed to be skin contaminants since they grew in the culture before the patient was connected to the machine. The Klebsiella bacteremia occwTed only once midway during a dialysis. Ten of the 17 patients (59 per cent) had at least one positive culture for Ps. aeruginosa some time during the study. Positive cultures were obtained only after the coils had been reused at least five times. At the sixth use only one of 22 dialyses (5 per cent) was associated with a positive culture for Pseudomonas, but by the tenth use this had increased to seven of 17 dialyses (41 per cent). The data from patients with positive blood cultures are summarized in Table II. Most were found at the end of dialysis. In only two patients did cultures positive for Pseudomonasdevelop midway through dialysis; cultures later in dialysis in each of these patients were also
May
1977
The
Amerlcan Journal of Medicine
Volume 62
673
PS. AERUGINOSA BACTEREMIA IN A DIALYSIS UNIT. II.-WAGNILD
TABLE
Patients
II
with Positive
ET AL.
Blood Cultures
Dialysate Blood _ _
Coil Use
When Positive*
1
9 10’
4 4
Pseudomonas Pseudomonas
2
9 101 101 8+ 4
4 4 4
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Klehsiella Staph. epidermidis Pseudomonas
7 7 NT 7 and 5
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas
7 and 5 7 and 5 7 7 and 5 7 and 5
Case No.
3 4 5
9 104 6 7
8 9 10 11
2. 3,4 3 1 4
9 10 8 9t
4 4 4 4
lot
2, 3.4
2 6+ 101 7*
1 4 4 4
._ ..__
positive.
Tvd
Organism
7
7
Diphtherolds Pseudomonas Pseudomonas Pseudomonas
’ Time during dialysis blood for cultures was drawn; 7 First number represents predominant immunotype - Last coil use
_..___
7 5
Pseudomonas Pseudomonas, Esch. coli Pseudomonas Pseudomonas Klebsiella Pseudomonas Pseudomonas, Esch. coli Pseudomonas
-
Organisms/ml
Systemic Symptoms
Before
After
IO3 lo2
lo7 lob
None None
10’ 10’ 10’ lo3 10’ 10’ 10’
lo1 10; lo5 los lob 10’ lo4
None None None None Muscle cramps None None
10’ 10’
lo3 lo4 lo= 10’ lo3
10) -
lo4
None None None None Chills, fever after dialysis None None None None
-
1, at start; 2. at 2 hours; 3, at 4 hours; 4, at end of dialysis. when two organisms were recovered; NT = nontypable.
Four
May 1977
Pseudomonas Pseudomonas, Esch. coli Pseudomonas Pseudomonas Pseudomonas Pseudomonas
2 and NT
coils which were used during dialyses associated with positive cultures were sterilized and reused. In each instance cultures of blood drawn at the end of the subsequent dialyses were also positive although cultures of blood obtained at the start of these dialyses were negative. The Pseudomonas immunotypes isolated most frequently from the blood were 7 and 5. Type 2 was detected only once. In four patients, two different immunotypes were recovered from the blood. The Pseudomonas isolated from each of the positive blood cultures was resistant to benzalkonium chloride. All were able to grow in the presence of 1,000 pg/ml of this chemical. In only one of the patients in this survey (Case 7) did systemic symptoms develop which could probably be related to the positive blood cultures. Although this patient’s temperature was normal during dialysis, she experienced chills and fever the evening following dialysis. Throughout the entire six weeks of the survey temperature readings over 1OO’F were recorded in only two patients, and blood cultures in both of these patients were negative. Dialysate Cultures. At the stat-t of dialysis, bacteria were present in all but eight of the 170 dialysates cultured with colony counts of 10’ to lo3 organisms/ml. Pseudomonas, Klebsiella and Escherichia coli were the most commonly isolated organisms. All dialysate
674
Predominant Organism
The American Journal of Medicine
Volume 62
samples obtained after dialysis were contaminated with 1O3to lo7 colonies/ml of the same organisms. No difference was apparent in the extent of bacterial contamination of the dialysates of patients with or without positive blood cultures. Coil Cultures. Forty-two coils were cultured after being used from one to 10 times. Bacteria were grown from all coils, 36 of which contained Ps. aeruginosa. Blood for cultures was obtained from patients during the use of 15 of these coils, and in five of these patients the cultures were positive for Pseudomonas. In each of these five, the same Pseudomonas immunotype was isolated from both the blood and coil. Cultures of tap water used to make the sterilizing benzalkonium chloride solution were negative. In addition, no Pseudomonas could be grown from MilliporeTm filters after filtering 4 liters of tap water. Forty-eight single use coils were filled with media following dialysis without undergoing prior cleaning and storage. Seven unused coils were similarly processed as controls. Results of these studies are presented in Table III. None of the seven unused control coils was contaminated with Pseudomonas although three did contain other organisms such as Staphylococcus epidermidis and Bacillus species. Thirty-two of the 48 single use coils (67 per cent) grew Ps. aeruginosa. Thirteen coils contained other organisms. This included three of the 32 containing Ps. aeruginosa plus an ad-
PS. AERUGINOSA
TABLE
III
Results
of Culture
of Single Use Coils No. Positive
Total No. Unused COIIS Single use coils * lmmunotypes 1, 5 (4). type (I).
7 48 recovered 6 (21,type
Pseudom. onas 0 32'
Other Organisms 3 13
No. Negative 4 6
included type 7 (18). type 2 (4). type 3, 7 (2),type 4, 6,7 (1) and type 4. 6
ditional 10 coils. Other bacteria consisted of Staphylococcus epidermidis, Bacillus species, Ps. cepacia, Flavobacterium and Xanthamonas. Type 7 Pseudomonas was the most common isolate, being found in 18 of the coils whereas six other types were present in from one to four of the coils. COMMENTS
Coils are reused in many dialysis centers as a means of curtailing the high cost of dialysis. This practice has been utilized for over eight years at our hospital without apparent detriment to patients. However, after the detection of Ps. aeruginosa bacteremia in four patients during a six month period, we undertook a systematic bacteriologic survey of the dialysis operation. The finding of silent Pseudomonas bacteremia during dialysis in 10 of 17 patients over a six week period was unexpected. Routine culture of the saline wash just prior to connecting patients to reutilized coils was always sterile. However, by incubating media-filled coils, it was found that all multiple and two thirds of single use coils were contaminated with P.s. aeruginosa, most likely from the dialysate. After five uses, the number of residual bacteria in the coil presumably became large enough in many instances to lead to bacteremia during dialysis. Thus the method of cleaning and sterilizing coils using benzalkonium chloride did not eradicate Pseudomonas. Bacteremia in patients undergoing hemodialysis is a relatively rare event despite the frequent occurrence of febrile episodes in this population [9,10]. Nevertheless, if bacteremia develops in patients undergoing dialysis, they are at increased risk for endocarditis and systemic infection because of the presence of fistulas or shunts and the decreased resistance to infection in uremia [11,12]. Cultures of blood obtained when patients experience chills and fever during dialysis are rarely positive [9,10]. In addition, Robinson and Rosen [lo] reported the same low frequency of positive cultures in concomitantly sampled asymptomatic and febrile patients. At present the majority of pyrexial reactions are presumed to be caused by endotoxin or other bacterial products
BACTEREMIA
IN A DIALYSIS
UNIT. II
-WAGNILD ET AL.
crossing the dialysis membrane [ 9,13,14]. However, the exact mechanism whereby endotoxin with a molecular weight of approximately 200,000 crosses the membrane is unknown but is presumably due to minute tears or breaks in the membrane. Raij et al. [9] demonstrated passage of endotoxin from the dialysate to blood compartment in two of six Kiil type dialyzers, and Malchesby et al. [ 151 found leaks at membrane wrinkles in the sealing area in 100 per cent of the nearly 50 coils tested. Because intracoil pressure is usually equal to or higher than the surrounding dialysis fluid pressure, passage of bacteria or their products into the blood compartment would be retarded. Nevertheless, pyrexial reactions have been noted to be proportional to the level of bacterial contamination of the dialysate [ 161, and one might expect that bacteria entering the blood would similarly increase. Direct passage across intact coil membranes, however, has not been demonstrated. Tierno et al. [ 171 were also unable to detect it immediately after coil rupture. It seems most likely that the coils in our study became contaminated during dialysis and not as a result of the resterilization procedure for several reasons. First, Pseudomonas could not be grown from the tap water used to make the benzalkonium chloride solution, and second, Pseudomonas could be cultured from most single use coils which had not undergone resterilization. These latter data strongly suggest that the coils became infected by a low level of bacterial crossover from the dialysate to blood compartment. However, other modes of entry, such as through the air vent in the drip chamber, cannot be absolutely excluded. The recovery of additional bacterial species other than Pseudomonas in both control and single use coils may have been due to occasional contamination during the procedure for culturing coils. However, contamination cannot explain all the data since the frequency of recovery of Ps. aeruginosa from single use coils was significantly greater than that from control coils, and different immunotypes of Ps. aeruginosa were recovered from coils processed on the same day. Pseudomonas was cultured from nearly all dialysate samples and was always present when blood cultures were positive. In one patient (Case 7), however, Pseudomonas was not the predominant organism. The number of Pseudomonas in the bath at the end of dialysis in patients with positive and negative blood cultures did not differ. Unfortunately, we did not save the organisms isolated from the coil compartment at the time of bacteremia and therefore cannot compare bath immunotypes with that found in the blood and coil. Certain strains or mutants of Pseudomonas resistant to quaternary ammonium compounds such as benzalkonium chloride have produced serious hospital-acquired infections [ 18,191. The Pseudomonas isolated
May 1977
The American Journal of Medicine
Volume 62
675
PS. AERUGINOSA
BACTEREMIA
IN A DIALYSIS
UNIT. I:.-WAGNILD
ET AL
from our patients were also resistant to benzalkonium chloride. It seems possible that continued buildup of these resistant bacteria occurred in reutilized coils, possibly in leukocyte or platelet clots [ 201. This then could have lead to shedding of bacteria during reuse. Another explanation might be that after multiple uses, small tears or cracks developed in the coil, thereby permitting passage of sufficient bacteria from the dialysate to cause bacteremia. This seems unlikely since the bacteremia would be expected to occur throughout rather than just at the end of dialysis. Pseudomonas were always present in the dialysate at the start of dialysis, although not in as high a concentration as at the end. In addition, only Pseudomonas were grown in the blood whereas other bacteria were also present in the dialysate. Finally, single use coils already contained Pseudomonas. Initial blood cultures at the time of the ensuing dialysis were always negative indicating that the bacteremia was not persistent. The level of virulence of these Pseudomonas organisms was probably low as evidenced by lack of systemic symptoms in nine
1. 2. 3. 4. 5.
6.
7.
8.
9. 10. 11. 12. 13.
676
Ahmad Ft. Goldsmith HJ: Automated diilyser rinsing machine. Dialysis and Transplantation 4: 29, 1975. Bilinsky B, Morris AJ: Hemodialysis coil reuse. A safe and economical new method. JAMA 218: 1808, 1971. Fawcett CK. Mangles MD: Reuse of the Gambro Lundia 17layer dialyrer. Dialysis and Transplantation. 3: 38. 1974. &II RP, Figuema JE: Haemodialysis cost reduction by artificial kidney storage. Br Med J 1: 788, 1970. Johnson CE, Dctaviano GN, Beirne 6). et al.: Cleaning, storage and repeated use of twin coil dialyzing units. JAMA 207: 2087, 1969. Uman SJ, Johnson CE, Beirne 61, et al.: Pseudomonas aeruginosa bacteremia In a dialysis unit. I. Recognition of cases, epidemiologic studies and attempts at control. Am J Med 62: 667, 1977. Fisher MW, Devlin HB, @tabasik FJ: New immunotype schema for pseudomonas eeruginosa based on protective antigens. J Bacterial 98: 835, 1969. Adair FW, Geflic SG, Gelzer J: Resistance of Pseudomonas to quaternary ammonium compounds. I. Growth in benzalkonium chloride solution. Appl Mlcrobiol 18: 299, 1969. Raij L, Shapiro FL, Michael AF: Endotoxemia in febrile reactions during hemodialysis. Kidney Int 4: 57, 1973. Robinson PJA, Rosen SM: Pyrexial reactlons during haemodialysis. Br Med J 1: 528, 1971. Leonard A, Raij L, Shapiro F: Bacterial endocardttis in regularly dialyzed patients. Kidney Int 4: 407, 1973. Montgomerie JZ, Kalmanson GM, Guze LB: Renal failure and infection. Medicine (Baltimore) 47: 1, 1968. Hindman SH, Favero MS, Carson LA, et al.: Pyrogenic reac-
May 1077
The American
Journal of Medicine
Volume 62
of the 10 patients. Studies in animals have also suggested decreased virulence of Pseudomonas organisms resistant to benzalkonium chloride [21]. Recently Kuehnel et al. [ 221 reported an acute outbreak of Pseudomonas cepacia bacteremia related to contaminated reused coils. They, too, used benzalkonium chloride as a sterilizing agent but only noted clinically apparent bacteremia when the coils were inadvertently left unrefrigerated for several days and then reused. Microbial contamination of the dialysate and its distribution systems is recognized as an area requiring constant surveillance. Technologic improvement in design and operation of dialysis machines is needed to lessen the high risk of infection in patients undergoing dialysis [ 16,231. Reused equipment should be monitored by culturing technics, such as incubation of media-filled coils. Because of the increased risk for infection inherent in coil resterilization, we have chosen to discontinue this practice in our hemodialysis program.
14.
15.
16.
17.
18. 19.
20.
21.
22.
23.
tions during haemodialysis caused by extramural endotoxin. Lancet 2: 732, 1975. Gazenfield-Gazit E, Eliahou HE: Endotoxin antibodies in patients on maintenance hemcdialysis. lsr J Med Sci 5: 1032, 1969. Malchesky PS, Surovy R, Kiraly R, et al.: Hidden hazards of coil dialysis. Proceedings of the Eleventh Congress of the European Dialysis and Transplant Association, Tel Aviv, Israel, 1974, p 48. Favero MS, Petersen NJ, Boyer KM, et al.: Microbial contamination of renal dialysis systems and associated health risks. Trans Am Sot Artif Intern Organs 10: 175, 1974. Tiirna PM Jr, Aboddy R: Risk of bacterial infections resulting from a blood leak during hemcdialysis. Nephron 8: 110, 1969. Bassett DJC: Common-source outbreaks. Proc R Sot Med 64: 980, 1971. Phillips I, Eykyn S, Curtis MA, et al.: Pseudomonas cepacia (Multivorans) septicaemia in an intensive-care unit. Lancet 1: 375, 1971. Mason R, Zucker W, Bilinsky R, et al.: Blood components deposited on dialysis membranes of used and reused coil hemodlalyzers (abstract). Kidney Int 8: 429, 1975. Adair FW, Liauw H, Geftic SG, et al.: Reduced virulence of Pseudomonas aeruginosa grown In the presence of benzalkonium chloride. J Clin Microbial 1: 175. 1975. Kuehnel E, Lundh H: Outbreak of Pseudomonas cepacia bacteremia related to contaminated reused coils. Dialysis and Transpbntation 5: 44, 1976. Blagg CR, Tenckhoff H: Mlcrobtal contamination of water used for hemodialysis. Nephron 15: 81, 1975.
to Reuse of Coils
JON P. WAGNILD. M.D. PETER MCDONALD, M.D.’ WILLIAM A. CRAIG, M.D. CAROLINE JOHNSON, B.A. MICHAEL HANLEY, M.D.’ STEPHEN J. UMAN. M.D.* V. RAMGOPAL. M.B.B.S. GREGORY J. BEIRNE, M.D. Madison. Wisconsin
Blood for culture was obtained over a six week period from 17 patients undergoing long-term hemodialysts. Bacteremia was detected during 18 of 201 dialyses. Blood drawn during fifleen of these dialyses contained Pseudomonas aen@osa. Ten of the 17 pabents (59 per cent) had a Pseudomonas bacteremia some time during the study. Only one patient was symptomatic. The frequency of positlve cultures was related to reuse of co&. No cuttues were positive until after the fifth use, but by the tenth use, 41 per cent of the dialyses were associated wtth bacteremia. All coils that were used repeatedly and 32 of 48 of those used only once, grew Ps. aeruginosa when filled wlth media and Incubated. This suggests that the coils were inoculated during dialysis and that benzalkonium chloride, the sterilizing agent, was unable to eradicate this organism. With repeated uses, the number of residual bacteria in the coil became large enough to cause detectable bacteremia during dialysis. Dialysis coils are reused in many centers to decrease
From the Renal and Infectious Disease Units of the Medical Service, Veterans Administration Hospital: and the Department of Medicine, University of Wisconsin Schccl of Medicine, Medii, Wisconsin. Requests for reprints should be addressed to Dr. Jon P. Wagnild. Department of Medicine. Veterans Administration Hospital. 2500 Overlook Terrace, Madison, Wisconsin 53705. Manuscript accepted August 24, 1976. ’ Present address: Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000. ’ Present address: Department of Medicine, Nephrology Service, University of Texas Health Sciences Center, Dallas, Texas 75235. t Present address: 6360 Wilshire Boulevard, Los Angeles, California 90048.
672
May 1977
the high cost of hemodialysis. Several different methods of cleaning and storing coils have been proposed, and reuse of up to 15 times without significant complications or impairment in clearance characteristics has been reported [i-5]. In our dialysis center we have reused coils for over eight years without apparent ill effects and with considerable savings [5]. Coils were cleaned and stored using benzalkonium chloride as the sterilizing agent. However, over a recent six month period Pseudomonas bacteremia developed in four of 20 patients in w center, and one dled from Pseudomonas endocarditis [6]. To determine the source of the bacteremia, we undertook a bacteriologic survey of the hemodialysis unit. This included personnel, dialysate. machines, delivery system, coils, sterilization technic and, finally, the systematic collection of blood culties throughout each dialysis on all patients over a six week period. In this report we present the results of the survey involving cultures of the blood, coil and dialysate. In a companion paper we deal with the microbiologic studies of dialysate, machines and personnel [ 6 I. METHODS Seventeen patients with a stable condiiion undergoing lonpterm hemodiilysis were surveyed. All were dialyzed via a percutaneous puncture of a forearm arteriovenous fistula. For the first four weeks of the study, blood for culture was obtained from all 17 patients at the start of dialysis and at 2 hour intervals throughout each dialysis. Blood samples were drawn from the arterial line. Pediatric blood culture tubes (Vacutainer Culture Tube, Gentec Hospital Supply) were used which required only 2 ml of blood and thereby limited tie
The American Journal of Mediclne
Volume 62
PS
AERUGINOSA
total amount of blood withdrawn per 6 hour dialysis to 8 ml. Blood pressure and oral temperature measurements were recorded at each sampling time. Dialysate samples obtained from the coil compartment before dialysis were cultured on dip slides (Uricult. Medical Technology Corp.): dialysate samples obtained after dialysis were cultured on both dip slides and trypticase soy agar plates. The study was then continued an additional two weeks during which blood for culture was drawn only at the end of dialysis. This was from a group of patients whose coils were reused more than five times. If a blood culture became positive during this latter period, the dialysis unit was notified, and coils were either discarded or filled with media and cultured. A description of the dialysis unit has already been given [S]. Briefly, it is an eight station unit dosigned primarily for long-term hemodialysis. Approximately 19 patients undergo dialysis two to three times per week using Travenol RSPrm artificial kidney machines. Either a central proportioning system or bulk tanks are used for dialysate delivery. Cuprophane membrane coil dialyzers were routinely reused up to 10 times. Machines are interchanged between patients except in those suspected of or known to have infection or hepatitis. Coils to be reused were cleaned at the end of dialysis according to the following procedure. Blood remaining in the coil was flushed out with a saline solution made from tap water. When clear, the coil was filled with a solution of benzalkonitrn chloride in tap water (1:750), and stored at 4°C in a pail containing the same solution. Immediately prior to use, the benzalkonium chloride was poured off and the outside surface of the coils rinsed. They were then flushed with 3 liters of sterile distilled water and 1 liter of heparinized saline solution. During the infusion of a second liter of heparinized saline sofution, the coils were pressurt+cbecked and primed. Samples for culture were routinely obtained from the saline effluent from the coil prior to connecting the patient for dialysis. Culture Methods. Blood cultures were vented and incubated at 37% they were examined at 24 and 48 hours, and at one and two weeks. Dip slides of dialysis fluid were also incubated at 37’%, and the density was read at 24 hours. Samples of dialysate were refrigerated at 4°C and cultured within 24 hours. The predominating organisms were then picked and identified. Coils were cultured as follows: Initially, single and multiple use coils were cleaned and stored in the usual fashion, and prepared several days later for use. Trypticase soy broth (TSB) was then drawn into the coils from a farge flask utilizing the blood perfusion pumps, the blood tubings were clamped and the coils incubated at 37’C. Because of possible contamination during the cleaning or sterilizing procedure, a group of single use unsterilized coils was taken to the laboratory following dialysis, filled with TSB, clamped and incubated at 37’C for up to 10 days. Samples were subcultured at intervals by needle aspiration from the tubing. Media was drained from the coil at 10 days and further subcultured if earlier samples were negative for Ps. aeruginosa. All positive cultures were identified in the Clinical Microbiology Laboratory. Two to 6 colonies were individually subcultured from each blood and coil culture positive for Ps. aeruginosa. lmmunotyping was performed on each isolate
TABLE
average
BACTEREMIA
IN A DIALYSIS
Summary
I
UNIT
II
WAGNILD
ET AL
of Blood Cultures Positive for
Pseudomonas
with
Reuse of Coils
.._ Number of Times Coils were Used Data Patients studied DlalYSf?t studled P0SltlVe cultures Dialyses positive PntVmts positive
1
2
3
4
5
6
7
8
9
10
16
15
14
17
16
15
16
15
15
14
22
24
19
19
18
73
23
21
16
II
0
0
0
0
0
1
1
2
4
0
0
0
0
0
5
4
10
25
41
0
0
0
I)
0
7
6
13
27
50
(no.) ino. 7
(no.) 1%) (%I
by the slide agglutination method utilizing the antibody system described by Fischer et al. [7] using seven type-specific antiserums provided by t-f. B. Devlin, Ph.D., Parke-Davis Laboratories, Detroit, Mich. Resistance of Pseudomonas isolates to benzalkonium chloride was determined by the method of Adair et al. [ 81. Resistance is defined as growth in the presence of 1,000 pg/ml of benzalkoniumchloride.
RESULTS Blood Cultures. Blood for culture was obtained from 17 patients undergoing a total of 20 1 dialyses (Table I). During 170 of these dialyses, blood samples were drawn at the start and at 2 hour intervals thereafter; in the remaining 31 dialyses, only two blood samples for cultures were obtained at the end of dialysis. When examined at 24 and 48 hours, almost all blood cultures were negative. Only after incubation for longer periods of time, generally three to seven days, was growth sufficient to be detected. By this time the coils had been reused or discarded. Positive blood cultures were obtained in 18 of the 201 dialyses (9 per cent). Ps. aeruginosa was present in 15, and diphtheroids, Staphylococcus epidermidis and Klebsiella species were each isolated once. The diphtheroids and Staphylococcus epidermidis were presumed to be skin contaminants since they grew in the culture before the patient was connected to the machine. The Klebsiella bacteremia occwTed only once midway during a dialysis. Ten of the 17 patients (59 per cent) had at least one positive culture for Ps. aeruginosa some time during the study. Positive cultures were obtained only after the coils had been reused at least five times. At the sixth use only one of 22 dialyses (5 per cent) was associated with a positive culture for Pseudomonas, but by the tenth use this had increased to seven of 17 dialyses (41 per cent). The data from patients with positive blood cultures are summarized in Table II. Most were found at the end of dialysis. In only two patients did cultures positive for Pseudomonasdevelop midway through dialysis; cultures later in dialysis in each of these patients were also
May
1977
The
Amerlcan Journal of Medicine
Volume 62
673
PS. AERUGINOSA BACTEREMIA IN A DIALYSIS UNIT. II.-WAGNILD
TABLE
Patients
II
with Positive
ET AL.
Blood Cultures
Dialysate Blood _ _
Coil Use
When Positive*
1
9 10’
4 4
Pseudomonas Pseudomonas
2
9 101 101 8+ 4
4 4 4
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Klehsiella Staph. epidermidis Pseudomonas
7 7 NT 7 and 5
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas
7 and 5 7 and 5 7 7 and 5 7 and 5
Case No.
3 4 5
9 104 6 7
8 9 10 11
2. 3,4 3 1 4
9 10 8 9t
4 4 4 4
lot
2, 3.4
2 6+ 101 7*
1 4 4 4
._ ..__
positive.
Tvd
Organism
7
7
Diphtherolds Pseudomonas Pseudomonas Pseudomonas
’ Time during dialysis blood for cultures was drawn; 7 First number represents predominant immunotype - Last coil use
_..___
7 5
Pseudomonas Pseudomonas, Esch. coli Pseudomonas Pseudomonas Klebsiella Pseudomonas Pseudomonas, Esch. coli Pseudomonas
-
Organisms/ml
Systemic Symptoms
Before
After
IO3 lo2
lo7 lob
None None
10’ 10’ 10’ lo3 10’ 10’ 10’
lo1 10; lo5 los lob 10’ lo4
None None None None Muscle cramps None None
10’ 10’
lo3 lo4 lo= 10’ lo3
10) -
lo4
None None None None Chills, fever after dialysis None None None None
-
1, at start; 2. at 2 hours; 3, at 4 hours; 4, at end of dialysis. when two organisms were recovered; NT = nontypable.
Four
May 1977
Pseudomonas Pseudomonas, Esch. coli Pseudomonas Pseudomonas Pseudomonas Pseudomonas
2 and NT
coils which were used during dialyses associated with positive cultures were sterilized and reused. In each instance cultures of blood drawn at the end of the subsequent dialyses were also positive although cultures of blood obtained at the start of these dialyses were negative. The Pseudomonas immunotypes isolated most frequently from the blood were 7 and 5. Type 2 was detected only once. In four patients, two different immunotypes were recovered from the blood. The Pseudomonas isolated from each of the positive blood cultures was resistant to benzalkonium chloride. All were able to grow in the presence of 1,000 pg/ml of this chemical. In only one of the patients in this survey (Case 7) did systemic symptoms develop which could probably be related to the positive blood cultures. Although this patient’s temperature was normal during dialysis, she experienced chills and fever the evening following dialysis. Throughout the entire six weeks of the survey temperature readings over 1OO’F were recorded in only two patients, and blood cultures in both of these patients were negative. Dialysate Cultures. At the stat-t of dialysis, bacteria were present in all but eight of the 170 dialysates cultured with colony counts of 10’ to lo3 organisms/ml. Pseudomonas, Klebsiella and Escherichia coli were the most commonly isolated organisms. All dialysate
674
Predominant Organism
The American Journal of Medicine
Volume 62
samples obtained after dialysis were contaminated with 1O3to lo7 colonies/ml of the same organisms. No difference was apparent in the extent of bacterial contamination of the dialysates of patients with or without positive blood cultures. Coil Cultures. Forty-two coils were cultured after being used from one to 10 times. Bacteria were grown from all coils, 36 of which contained Ps. aeruginosa. Blood for cultures was obtained from patients during the use of 15 of these coils, and in five of these patients the cultures were positive for Pseudomonas. In each of these five, the same Pseudomonas immunotype was isolated from both the blood and coil. Cultures of tap water used to make the sterilizing benzalkonium chloride solution were negative. In addition, no Pseudomonas could be grown from MilliporeTm filters after filtering 4 liters of tap water. Forty-eight single use coils were filled with media following dialysis without undergoing prior cleaning and storage. Seven unused coils were similarly processed as controls. Results of these studies are presented in Table III. None of the seven unused control coils was contaminated with Pseudomonas although three did contain other organisms such as Staphylococcus epidermidis and Bacillus species. Thirty-two of the 48 single use coils (67 per cent) grew Ps. aeruginosa. Thirteen coils contained other organisms. This included three of the 32 containing Ps. aeruginosa plus an ad-
PS. AERUGINOSA
TABLE
III
Results
of Culture
of Single Use Coils No. Positive
Total No. Unused COIIS Single use coils * lmmunotypes 1, 5 (4). type (I).
7 48 recovered 6 (21,type
Pseudom. onas 0 32'
Other Organisms 3 13
No. Negative 4 6
included type 7 (18). type 2 (4). type 3, 7 (2),type 4, 6,7 (1) and type 4. 6
ditional 10 coils. Other bacteria consisted of Staphylococcus epidermidis, Bacillus species, Ps. cepacia, Flavobacterium and Xanthamonas. Type 7 Pseudomonas was the most common isolate, being found in 18 of the coils whereas six other types were present in from one to four of the coils. COMMENTS
Coils are reused in many dialysis centers as a means of curtailing the high cost of dialysis. This practice has been utilized for over eight years at our hospital without apparent detriment to patients. However, after the detection of Ps. aeruginosa bacteremia in four patients during a six month period, we undertook a systematic bacteriologic survey of the dialysis operation. The finding of silent Pseudomonas bacteremia during dialysis in 10 of 17 patients over a six week period was unexpected. Routine culture of the saline wash just prior to connecting patients to reutilized coils was always sterile. However, by incubating media-filled coils, it was found that all multiple and two thirds of single use coils were contaminated with P.s. aeruginosa, most likely from the dialysate. After five uses, the number of residual bacteria in the coil presumably became large enough in many instances to lead to bacteremia during dialysis. Thus the method of cleaning and sterilizing coils using benzalkonium chloride did not eradicate Pseudomonas. Bacteremia in patients undergoing hemodialysis is a relatively rare event despite the frequent occurrence of febrile episodes in this population [9,10]. Nevertheless, if bacteremia develops in patients undergoing dialysis, they are at increased risk for endocarditis and systemic infection because of the presence of fistulas or shunts and the decreased resistance to infection in uremia [11,12]. Cultures of blood obtained when patients experience chills and fever during dialysis are rarely positive [9,10]. In addition, Robinson and Rosen [lo] reported the same low frequency of positive cultures in concomitantly sampled asymptomatic and febrile patients. At present the majority of pyrexial reactions are presumed to be caused by endotoxin or other bacterial products
BACTEREMIA
IN A DIALYSIS
UNIT. II
-WAGNILD ET AL.
crossing the dialysis membrane [ 9,13,14]. However, the exact mechanism whereby endotoxin with a molecular weight of approximately 200,000 crosses the membrane is unknown but is presumably due to minute tears or breaks in the membrane. Raij et al. [9] demonstrated passage of endotoxin from the dialysate to blood compartment in two of six Kiil type dialyzers, and Malchesby et al. [ 151 found leaks at membrane wrinkles in the sealing area in 100 per cent of the nearly 50 coils tested. Because intracoil pressure is usually equal to or higher than the surrounding dialysis fluid pressure, passage of bacteria or their products into the blood compartment would be retarded. Nevertheless, pyrexial reactions have been noted to be proportional to the level of bacterial contamination of the dialysate [ 161, and one might expect that bacteria entering the blood would similarly increase. Direct passage across intact coil membranes, however, has not been demonstrated. Tierno et al. [ 171 were also unable to detect it immediately after coil rupture. It seems most likely that the coils in our study became contaminated during dialysis and not as a result of the resterilization procedure for several reasons. First, Pseudomonas could not be grown from the tap water used to make the benzalkonium chloride solution, and second, Pseudomonas could be cultured from most single use coils which had not undergone resterilization. These latter data strongly suggest that the coils became infected by a low level of bacterial crossover from the dialysate to blood compartment. However, other modes of entry, such as through the air vent in the drip chamber, cannot be absolutely excluded. The recovery of additional bacterial species other than Pseudomonas in both control and single use coils may have been due to occasional contamination during the procedure for culturing coils. However, contamination cannot explain all the data since the frequency of recovery of Ps. aeruginosa from single use coils was significantly greater than that from control coils, and different immunotypes of Ps. aeruginosa were recovered from coils processed on the same day. Pseudomonas was cultured from nearly all dialysate samples and was always present when blood cultures were positive. In one patient (Case 7), however, Pseudomonas was not the predominant organism. The number of Pseudomonas in the bath at the end of dialysis in patients with positive and negative blood cultures did not differ. Unfortunately, we did not save the organisms isolated from the coil compartment at the time of bacteremia and therefore cannot compare bath immunotypes with that found in the blood and coil. Certain strains or mutants of Pseudomonas resistant to quaternary ammonium compounds such as benzalkonium chloride have produced serious hospital-acquired infections [ 18,191. The Pseudomonas isolated
May 1977
The American Journal of Medicine
Volume 62
675
PS. AERUGINOSA
BACTEREMIA
IN A DIALYSIS
UNIT. I:.-WAGNILD
ET AL
from our patients were also resistant to benzalkonium chloride. It seems possible that continued buildup of these resistant bacteria occurred in reutilized coils, possibly in leukocyte or platelet clots [ 201. This then could have lead to shedding of bacteria during reuse. Another explanation might be that after multiple uses, small tears or cracks developed in the coil, thereby permitting passage of sufficient bacteria from the dialysate to cause bacteremia. This seems unlikely since the bacteremia would be expected to occur throughout rather than just at the end of dialysis. Pseudomonas were always present in the dialysate at the start of dialysis, although not in as high a concentration as at the end. In addition, only Pseudomonas were grown in the blood whereas other bacteria were also present in the dialysate. Finally, single use coils already contained Pseudomonas. Initial blood cultures at the time of the ensuing dialysis were always negative indicating that the bacteremia was not persistent. The level of virulence of these Pseudomonas organisms was probably low as evidenced by lack of systemic symptoms in nine
1. 2. 3. 4. 5.
6.
7.
8.
9. 10. 11. 12. 13.
676
Ahmad Ft. Goldsmith HJ: Automated diilyser rinsing machine. Dialysis and Transplantation 4: 29, 1975. Bilinsky B, Morris AJ: Hemodialysis coil reuse. A safe and economical new method. JAMA 218: 1808, 1971. Fawcett CK. Mangles MD: Reuse of the Gambro Lundia 17layer dialyrer. Dialysis and Transplantation. 3: 38. 1974. &II RP, Figuema JE: Haemodialysis cost reduction by artificial kidney storage. Br Med J 1: 788, 1970. Johnson CE, Dctaviano GN, Beirne 6). et al.: Cleaning, storage and repeated use of twin coil dialyzing units. JAMA 207: 2087, 1969. Uman SJ, Johnson CE, Beirne 61, et al.: Pseudomonas aeruginosa bacteremia In a dialysis unit. I. Recognition of cases, epidemiologic studies and attempts at control. Am J Med 62: 667, 1977. Fisher MW, Devlin HB, @tabasik FJ: New immunotype schema for pseudomonas eeruginosa based on protective antigens. J Bacterial 98: 835, 1969. Adair FW, Geflic SG, Gelzer J: Resistance of Pseudomonas to quaternary ammonium compounds. I. Growth in benzalkonium chloride solution. Appl Mlcrobiol 18: 299, 1969. Raij L, Shapiro FL, Michael AF: Endotoxemia in febrile reactions during hemodialysis. Kidney Int 4: 57, 1973. Robinson PJA, Rosen SM: Pyrexial reactlons during haemodialysis. Br Med J 1: 528, 1971. Leonard A, Raij L, Shapiro F: Bacterial endocardttis in regularly dialyzed patients. Kidney Int 4: 407, 1973. Montgomerie JZ, Kalmanson GM, Guze LB: Renal failure and infection. Medicine (Baltimore) 47: 1, 1968. Hindman SH, Favero MS, Carson LA, et al.: Pyrogenic reac-
May 1077
The American
Journal of Medicine
Volume 62
of the 10 patients. Studies in animals have also suggested decreased virulence of Pseudomonas organisms resistant to benzalkonium chloride [21]. Recently Kuehnel et al. [ 221 reported an acute outbreak of Pseudomonas cepacia bacteremia related to contaminated reused coils. They, too, used benzalkonium chloride as a sterilizing agent but only noted clinically apparent bacteremia when the coils were inadvertently left unrefrigerated for several days and then reused. Microbial contamination of the dialysate and its distribution systems is recognized as an area requiring constant surveillance. Technologic improvement in design and operation of dialysis machines is needed to lessen the high risk of infection in patients undergoing dialysis [ 16,231. Reused equipment should be monitored by culturing technics, such as incubation of media-filled coils. Because of the increased risk for infection inherent in coil resterilization, we have chosen to discontinue this practice in our hemodialysis program.
14.
15.
16.
17.
18. 19.
20.
21.
22.
23.
tions during haemodialysis caused by extramural endotoxin. Lancet 2: 732, 1975. Gazenfield-Gazit E, Eliahou HE: Endotoxin antibodies in patients on maintenance hemcdialysis. lsr J Med Sci 5: 1032, 1969. Malchesky PS, Surovy R, Kiraly R, et al.: Hidden hazards of coil dialysis. Proceedings of the Eleventh Congress of the European Dialysis and Transplant Association, Tel Aviv, Israel, 1974, p 48. Favero MS, Petersen NJ, Boyer KM, et al.: Microbial contamination of renal dialysis systems and associated health risks. Trans Am Sot Artif Intern Organs 10: 175, 1974. Tiirna PM Jr, Aboddy R: Risk of bacterial infections resulting from a blood leak during hemcdialysis. Nephron 8: 110, 1969. Bassett DJC: Common-source outbreaks. Proc R Sot Med 64: 980, 1971. Phillips I, Eykyn S, Curtis MA, et al.: Pseudomonas cepacia (Multivorans) septicaemia in an intensive-care unit. Lancet 1: 375, 1971. Mason R, Zucker W, Bilinsky R, et al.: Blood components deposited on dialysis membranes of used and reused coil hemodlalyzers (abstract). Kidney Int 8: 429, 1975. Adair FW, Liauw H, Geftic SG, et al.: Reduced virulence of Pseudomonas aeruginosa grown In the presence of benzalkonium chloride. J Clin Microbial 1: 175. 1975. Kuehnel E, Lundh H: Outbreak of Pseudomonas cepacia bacteremia related to contaminated reused coils. Dialysis and Transpbntation 5: 44, 1976. Blagg CR, Tenckhoff H: Mlcrobtal contamination of water used for hemodialysis. Nephron 15: 81, 1975.