Quantitative Microbiological Monitoring of Hemodialysis Fluids: Evaluation of Methods and Demonstration of Lack of

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1982, p Vol. 16, No /82/ $02.00/0 Copyright C 1982, American Society for Microbiology Quantitative Microbiological Monitoring of Hemodialysis Fluids: Evaluation of Methods and Demonstration of Lack of Test Relevance in Single-Pass Hemodialysis Machines with Automatic Dialysate Proportioning with Reverse Osmosis- Treated Tap Water GARY V. DOERN,l 2* BRENDA E. BROGDEN,1 JUDITH D. DiFEDERICO,' JANE E. EARLS,1 AND MARY L. QUINN' Clinical Microbiology Laboratories' and Division of Infectious Disease,2 University of Massachusetts Medical Center, Worcester, Massachusetts Received 23 April 1982/Accepted 2 August 1982 Two methods for estimating the quantity of microorganisms present in hemodialysis fluid, a blood agar surface-spread plate method and a total-count water tester device impregnated with modified standard plate count agar (Millipore Corp., Bedford, Mass.), were evaluated. Both methods exhibited comparable precision; however, colony counts obtained with the total-count water tester were consistently and unacceptably low. The need for routine quantitative microbiological monitoring of hemodialysis fluids such as that recommended by the American Public Health Association was not supported by the results of this study. Such testing was not of value in predicting untoward reactions for patients undergoing hemodialysis, nor did quantitative testing of hemodialysis fluids identify the buildup of potentially hazardous levels of contamination within hemodialysis systems. Finally, the kinds of organisms found in hemodialysis systems, i.e., gram-negative water-borne bacilli, were elucidated. Patients undergoing hemodialysis are at risk for the development of endotoxin-mediated pyrogenic reactions and gram-negative bacteremia with sepsis (3, 4, 6). These untoward side effects are thought to be related to the presence of excessive levels of gram-negative bacillary contamination of dialysis fluid used in hemodialysis treatment systems (2-4). This cause-and-effect relationship has led to the establishment of microbiological guidelines pertaining to allowable levels of contamination in dialysis fluid, recommendations regarding frequency of monitoring, and a list of methods considered to be acceptable for the performance of quantitative testing (1) Ṫhe intent of the present study was to evaluate two quantitative testing methods, to define the kinds of organisms usually found in hemodialysis systems, and to assess the value of such testing. MATERIALS AND METHODS Dialysis fluid specimen collection. A total of 52 consecutive hemodialysis treatments performed on two hemodialysis machines (23 on machine A and 29 on machine B) were investigated over a 63-day period. Both machines were single-pass Single Patient System hemodialysis instruments (Extracorporeal, Inc., King 1025 of Prussia, Pa.) equipped with capillary cartridge dialyzers. By using asceptic techniques, 50 ml of dialysis fluid was collected just before and immediately after each dialysis treatment. The predialysis sample represented fluid that had been proportioned in the dialysis machine to the appropriate ionic strength from dialysate concentrate (Diasol 34 Standard, Travenol Laboratories, Deerfield, Ill.) and tap water previously treated by reverse osmosis. Tap water treated by reverse osmosis flowed continuously on demand from a reverse osmosis treatment system into the proportioner. The postdialysis specimen was collected within 3 min of the completion of a dialysis treatment. All samples were transported directly to the laboratory and processed within 5 min. During the course of this study, both machines were thoroughly cleaned after each treatment with 5.25% sodium hypochlorite. In addition, each was disinfected monthly with a solution of 3.7% formaldehyde. The dialysis membrane cartridge unit was replaced after each treatment. Microbiological analyses. All analyses were performed in duplicate. Dialysis fluid samples were first agitated to ensure an even distribution of microorganisms within the specimen. By using a sterile 1.0-ml graduated plastic pipette, 0.1 ml of fluid was transferred to the surface of a 5.0% defibrinated sheep blood agar plate and spread evenly across the plate surface (blood agar surface-spread plate procedure [BASP]). By using a sterile 10.0-ml graduated plastic pipette, the plastic sample reservoir of a total-count water tester (TCWT) impregnated with modified stan-

2 1026 DOERN ET AL. J. CLIN. MICROBIOL. 4 fornmakdhyde clrmaidengi formaldehyde clearnng cbasng cleating t4It IIACK04 A] s Day of Study FIG. 1. Results of quantitative microbiological analyses of predialysis (open bars) and postdialysis (hatched bars) fluid samples collected before and after 52 different hemodialysis treatments on two machines. Formaldehyde disinfection of both machines was undertaken at the times indicated by the arrows. dard plate count medium (Millipore Corp., Bedford, Mass.) was filled to the desired level as indicated by the fill line on the reservoir (i.e., approximately 10 ml of fluid), and the paddle containing filter and medium was inserted into the fluid. The device was agitated vigorously and then held in a vertical position for 1 min. The paddle was removed from the reservoir, the residual dialysis fluid was emptied, and the paddle was reinserted. The empty plastic reservoir thus served as an incubation chamber. The plates and testers were incubated for 48 h at 35 C in room air and examined for the presence of colonies. The number of colonies present on the sheep blood agar plates and on the TCWT were multiplied by 10 and 1, respectively, as an estimate of the colony-forming units (CFU) per milliliter of original sample. The Gram smear morphology of unique colony types was determined, and organisms present in quantities of >200 CFU/ml were identified according to conventional criteria. RESULTS The precision of two methods, a BASP and a TCWT, as means for determining the quantity of microorganisms in hemodialysis fluid was investigated. Pre- and postdialysis fluid samples were obtained from 52 different hemodialysis treatments. All samples were tested in duplicate by both methods. In 96 of 104 comparisons (92.3%) of the BASP, comparable results, defined as variation of <25% in colony counts, were obtained. Minor discrepancies, i.e., variations of 25 to 100%, were obtained with five (4.8%) of the duplicate BASP analyses. Major discrepancies, defined as >100% variation, were obtained in three (2.9%) instances. In no case was a difference of greater than 1 log noted between duplicate colony counts. In 93 of 104 comparisons (89.4%) of the TCWT, comparable results were obtained. Comparable results with the TCWT were defined as no growth on both testers (69 cases), variation of <25% when growth was detected on both testers (9 cases), or no growth on one tester with 1 to 10 colonies on the other (15 cases). In seven (6.7%) instances, minor discrepancies were noted. Minor discrepancies with the TCWT were defined as variation of 25 to 100% when growth was detected on both testers (four cases) or no growth on one tester with 11 to 200 colonies on the other (three cases). Major discrepancies with this method were observed in 4 of the 104 comparisons (3.8%). Major discrepancies were defined as >100% variation when growth was detected on both testers (three cases) or no growth on one tester with >200 colonies on the other (one case). A comparison of the results of colony counts obtained with the BASP and the TCWT on these 104 samples was made. All colony counts represent the mean of duplicate analyses. In cases in which one of the duplicate analyses with the TCWT yielded no growth whereas organisms were recovered from the other, the colony count from the latter was considered representative of that sample. In 56 samples containing 10 to 200 CFU/ml as determined by the BASP, no growth was observed in 49 (87.5%) cases with the TCWT, 1 to 10 CFU/ml was observed in 6 (10.7%) cases, and 11 to 200 CFU/ml was observed in 1 (1.8%) case. In 45 samples containing 210 to 2,000 CFU/ml with the BASP, 23 (51.1%) yielded no growth with the TCWT, 1 to 10 CFU/ ml was recovered in 11 (24.4%) cases, 11 to 200 CFU/ml was recovered in 10 (22.2%) cases, and >200 CFU/ml was recovered in 1 (2.2%) case. All three samples with >2,000 CFU/ml based on the results of the BASP yielded no growth with the TCWT. The 52 different hemodialysis treatments investigated in this study were conducted with two machines (A and B). A total of 23 treatments were conducted with machine A and 29 with machine B. Based on the results of the mean of duplicate analyses with the BASP, 43 (82.7%) of the 52 predialysis fluid samples contained a total of 10 to 200 CFU/ml (Fig. 1). The remaining nine

3 VOL. 16, 1982 TABLE 1. Organisms recovered from 52 pre- and posthemodialysis fluid samples Frequency observed in fluid No. of different samples taken: organisms recovered Predialysisa Postdialysisb a Of the 107 isolates recovered from predialysis fluid samples, 3 were yeasts, 4 were gram-positive cocci, and 100 were gram-negative bacilli. b Of the 140 isolates recovered from postdialysis fluid samples, 2 were gram-positive bacilli, 5 were yeasts, 7 were gram-positive cocci, and 124 were gram-negative bacilli. predialysis fluid samples (17.3%) contained a total of 210 to 2,000 CFU/ml. The number of different organisms recovered in any quantity from these 52 predialysis fluid samples is shown in Table 1. Fourteen organisms, all gram-negative bacilli, were present in quantities of >200 CFU/ml and were identified (Table 2). Among the 52 postdialysis fluid samples, 13 (25%) contained a total of 10 to 200 CFU/ml, 36 (69.2%) contained a total of 210 to 2,000 CFU/ ml, and 3 (5.8%) contained a total of >2,000 CFU/ml (Fig. 1). The number of different organisms recovered in any quantity from these 52 postdialysis fluid samples is shown in Table 1. Fifty-one organisms were present in quantities of >200 CFU/ml and were identified (Table 2). With the exception of one strain of Torulopsis glabrata, all were gram-negative bacilli. In 44 of 52 paired samples (84.6%), the postdialysis fluid contained greater quantities of organisms than the predialysis fluid (Fig. 1). In 30 (69.8%) of the 43 cases in which the predialysis samples contained 10 to 200 CFU/ml, the postdialysis specimen contained 210 to 2,000 CFU/ ml. In three (33.3%) of the nine cases in which 210 to 2,000 CFU/ml was recovered from predialysis samples, >2,000 CFU/ml was found in postdialysis fluid. In 6 of 52 paired samples (15.4%), the quantity of organisms recovered from pre- and postdialysis fluid was essentially the same. In no case was the quantity of organisms found in postdialysis fluid less than that recovered from predialysis fluid. Furthermore, in all 13 cases in which organisms were present in predialysis fluid in quantities of >200 CFU/ml and thus were identified, the same organism was present in postdialysis fluid at comparable (2 cases) or greater (11 cases) quantities. During the 2-month course of this study, none of the 52 patients who had undergone hemodialysis treatment on either machine suffered an HEMODIALYSIS FLUID TESTING 1027 untoward reaction. No discernable trend in dialysis fluid contamination was evident with either machine over the 63-day period of this study, either day to day, week to week, or month to month (Fig. 1). Lower colony counts were frequently followed by higher counts and vice versa. In all three instances in which postdialysis fluid samples contained >2,000 CFU/ml, postdialysis fluid samples obtained from the treatment immediately preceding it had colony counts of <200 CFU/ ml. In one case, the sample obtained from the treatment immediately after that showing the high colony count contained <200 CFU/ml. In the other two cases, <2,000 CFU/ml was recovered. Furthermore, no discernable trend in contamination was observed immediately after formaldehyde disinfection of either machine. In some instances, lower colony counts were observed; in other instances, higher counts were obtained. DISCUSSION Patients undergoing hemodialysis are at risk for the development of pyrogenic reactions and frank gram-negative sepsis (3, 4, 6). Pyrogenic reactions, usually characterized by fever, chills, and hypotension, generally become manifest within 1 to 5 h after the onset of dialysis (4) and are directly related to excessive levels of gramnegative bacilli present in dialysis fluids (2-4). Pyrogenic reactions are usually self-limiting, with abatement of symptoms and signs within minutes to a few hours after cessation of the dialysis treatment (4). The pathogenesis of such reactions is thought to include the release of endotoxin from gram-negative bacteria into dial- TABLE 2. Organisms present in quantities of greater than 200 CFU/ml in hemodialysis fluid No. of times recovered from Organism fluid taken: Pre- Postdialysis dialysis Flavobacterium meningosepticum 4 11 Pseudomonas aeruginosa 3 9 Achromobacter xylosoxidans 1 4 Alcaligenes faecalis 1 4 Flavobacterium odoratum 3 Chromobacterium violaceum 1 3 Pseudomonas fluorescens 3 Pseudomonas sp. (VA-1) 1 3 Pseudomonas sp. (VE-1) 1 2 Pseudomonas cepacia 1 2 Pseudomonas testosteroni 1 2 Pseudomonas putida 1 Pseudomonas pickettii 1 Flavobacterium sp. (IIb) 1 Achromobacter sp. (Vd) 1 Torulopsis glabrata 1

4 1028 DOERN ET AL. ysis fluid, with the passage of endotoxin across dialysis membranes into the patient's blood (5, 7-9). Gram-negative bacteremia leading to sepsis is also related to the presence of gramnegative bacilli in dialysis fluid. Organisms may pass directly through intact dialysis membranes when present in large numbers (10) or may gain entrance into the patient's blood through leaks in the membrane or the apparatus that houses it (12). Because of the ubiquitous nature of certain gram-negative bacilli in water used to prepare hemodialysis fluid, and because of the likely amplification of bacterial numbers in such fluid during dialysis treatment, guidelines pertaining to the minimum allowable levels of contamination in fluids used for hemodialysis have been developed by the American Public Health Association (1). Total viable counts in water used to prepare dialysis fluid should not exceed 200/ml. Total viable counts in dialysis fluid collected at the end of a hemodialysis treatment should not exceed 2,000/ml. In the absence of indications for more frequent sampling, samples for monitoring purposes should be collected at least monthly. Several methods for quantitating microorganisms in fluid are considered to be acceptable. The intent of the present study was fourfold. (i) The precision and utility of two methods which are considered to be acceptable for performing hemodialysis fluid monitoring, the BASP and the TCWT, were investigated. (ii) An attempt was made to define the kinds of organisms most likely to be found in dialysis fluids. (iii) Amplification during dialysis of contamination present in fluids before dialysis was investigated. (iv) An attempt was made to address the relevance of quantitative microbiological monitoring of hemodialysis fluids. The BASP and the TCWT possessed comparable levels of precision based on the results of duplicate analyses of 104 samples of dialysis fluid; however, the TCWT uniformly produced significantly lower colony counts than the BASP. The reasons for this discrepancy are unclear. The TCWT consists of a self-contained filtration-incubation device in which 1.0 ml of fluid sample is drawn through a tm-poresize Millipore filter onto an absorbent filter pad impregnated with dehydrated growth medium (R. A. Cotton, K. J. Sladek, and B. I. Sohn, Abstr. World Congr. Env. Med. Biol., Paris, 1974). Rehydrated growth medium then provides nutrients which support the development of colonies from microorganisms trapped on the membrane surface. One possible explanation for the consistently low colony counts obtained with the TCWT in this study is that bacteria present in dialysis fluid did not stick to the 0.45-,um-pore-size membrane, and thus, even after J. CLIN. MICROBIOL. rehydration of the growth Medium, colonies could not develop. Other possib"iities are that the nutrient filter pad did not absorb the desired 1.0-ml amount of fluid through the membrane or that incubation of the TCWT for 48 h as recommended by the manufacturer is inadequate to permit the development of visible colonies of the kinds of bacteria present in dialysis fluid specimens. This would seem unlikely since in several instances during this study, TCWT devices which had yielded no growth after 48 h of incubation were reincubated for as long as 120 h with no change in the results. Finally, it is possible that the m-spc medium used in the TCWT, a modification of standard plate count agar (11), did not support growth of the kinds of bacteria present in dialysis fluid specimens which were detectable on the sheep blood agar medium used with the BASP. In any case, the TCWT in its present form should not be considered reliable for estimating microbial contamination of dialysis fluid. The organisms found in dialysis fluid in the.highest concentrations both before and after dialysis were generally gram-negative bacilli known to be found in water. Flavobacterium meningosepticum and Pseudomonas aeruginosa were observed most frequently in large numbers; however, other species of Flav,obacterium and Pseudomonas as well as species of Achromobacter, Alcaligenes, and Chromobacterium were also recovered. Torulopis glabrata was recovered once in quantities of >200/ml. With this exception, gram-positive cocci and yeasts, when present, were always found in low concentrations. Pre- and postdialysis fluids were sampled in this study just before and immediately after dialysis, respectively. The predialysis fluid consisted of dialysate obtained after machine proportioning of dialysate concentrate with tap water that had been treated by reverse osmosis. This represents a departure from the recommended practice of sampling tap water before it is purified and mixed with concentrated dialysate. Since the system used to purify water in this study was reverse osmosis, which significantly reduces bacterial counts, and since sampling of tap water does not address potential additional contamination that can arise from either contaminated dialysate concentrate or the proportioning procedure, we felt that sampling of predialysis fluid as conducted in this study would be a more accurate reflection of the levels of contamination to which patients would be exposed during the early stages of the dialysis treatment. Furthermore, by comparing pre- and postdialysis culture results, the effect of the dialysis treatment alone on amplification of predialysis fluid contamination, as well as addition-

5 VOL. 16, 1982 HEMODIALYSIS FLUID TESTING 1029 al new contamination, could be determined. In this manner we hoped to address what would seem to be germane to the problem of untoward patient reactions, that is, contamination of dialysis fluid during dialysis, both at the onset and at the end of treatment. In 84.6% of the cases, the total level of contamination present in predialysis fluid was amplified during treatment. In all three cases in which the postdialysis counts were higher than proposed allowable levels (i.e., >2,000/ml), the predialysis counts were >200/ml. However, in six instances in which predialysis counts were >200/ml, the postdialysis counts, although increased, remained <2,000/ml. As demonstrated in a previous study (3), there was no consistent pattern in the magnitude of the amplification of counts during dialysis. It was observed, however, that predialysis counts of <200/ml were never associated with postdialysis counts above the recommended allowable levels. A total of 107 different organisms were recovered from predialysis samples, whereas 140 isolates were found in postdialysis samples. It is clear from these observations that new contamination did occur during dialysis treatment, but that this new contamination was usually not of a magnitude sufficient to raise postdialysis counts above the recommended allowable levels. The rationale behind quantitative microbiological monitoring of hemodialysis fluids is that such testing will identify levels of microbial contamination in hemodialysis systems which may pose a threat to patients undergoing hemodialysis treatment. The guidelines that have been established regarding unacceptable levels of contamination both in water used to prepare dialysate (>200 organisms per ml) and in dialysis fluid collected at the end of treatment (>2,000 organisms per ml) are predicated on estimates of the minimum concentration of organisms that, if present, could potentially lead to pyrogenic reactions or bacteremia. Monitoring of hemodialysis fluids is not intended as a means for predicting untoward side effects for an individual patient who has been treated with the actual fluid that has been tested, since the culture results for an individual sample would not be available until long after patient reactions had become manifest. Rather, the intent of monitoring is to identify the buildup of unacceptable levels of contamination within hemodialysis systems so that corrective action may be taken to avoid potential problems. Based on the results of the present study, it would seem that microbiological monitoring of hemodialysis fluids does not accomplish this goal. No pattern of contamination was evident when sampling of consecutive hemodialysis treatments was undertaken over a 2-month period. If this is so, then it would be unlikely that less frequent sampling, such as that recommended by the American Public Health Association (i.e., once monthly), would be effective in identifying contamination buildup. For this reason, the clinical utility of routine microbiological monitoring of hemodialysis fluids must be questioned. Given a rigorous program of cleaning, disinfection, and equipment maintenance, such monitoring would seem unnecessary, particularly in view of the emphasis on cost containment that presently exists in hospital microbiology laboratories. This recommendation pertains only to single-pass hemodialysis machines which utilize dialysis fluid that is machine proportioned from dialysate concentrate and reverse osmosis-treated tap water as described in this study. Routine microbiological monitoring of other hemodialysis systems, in the absence of additional studies, should be conducted according to published guidelines (1). ACKNOWLEDGMENTS We thank the Millipore Corp. for supplying the TCWTs used in this study. In addition, we appreciate the cooperation of Nancy Owens and the staff of the hemodialysis unit of the University of Massachusetts Medical Center. Finally, we acknowledge the excellent secretarial assistance of Annmarie McDonough. LITERATURE CITED 1. Committee on Microbial Contamination of Surfaces Microbiologic guidelines for hemodialysis systems. Health Lab. Sci. 15: Dawids, S. G., and R. Vejlsgaard Bacteriological and clinical evaluation of different dialysate delivery systems. Acta. Med. Scand. 199: Favero, M. S., N. J. Peterson, K. M. Boyer, L. A. Carson, and W. W. Bond Microbial contamination of renal dialysis systems and associated risks. Trans. Am. Soc. Artif. Intern. Organs 20: Favero, M. S., N. J. Petersen, L. A. Carson, W. W. Bond, and S. H. Hindman Gram negative water bacteria in hemodialysis systems. Health Lab. Sci. 12: Gazenfeldt-Gazit, E., and H. E. Eliahou Endotoxin antibodies in patients on maintenance hemodialysis. Isr. J. Med. Sci. 5: Hindman, S. H., M. S. Favero, L. A. Carson, N. J. Petersen, L. B. Schonberger, and J. T. Solano Pyrogenic reactions during hemodialysis caused by extramural endotoxin. Lancet ii: Jones, D. M., B. M. Tobin, G. R. Harlow, and A. J. Ralston Bacteriological studies of the modified Kiil dialyser. Br. Med. J. 3: Kidd, E. E Bacterial contamination of dialysing fluid of artificial kidney. Br. Med. J. 1: Raij, L., F. L. Shapiro, and A. F. Michael Endotoxemia in febrile reactions during hemodialysis. Kidney Int. 4: Schultz, J. S., and P. Gerhardt Dialysis culture of microorganisms: design, theory, and results. Bacteriol. Rev. 33: Taylor, R. H., and E. E. Geldreich A new membrane filter procedure for bacterial counts in potable water and swimming pool samples. J. Am. Well Water Assoc. 73: Tierno, P. M., and R. Aboody Risk of bacterial infection resulting from a blood leak during hemodialysis. Nephron 6: