Dialysis Fluid Samples Research Articles

Sanitary Waters: Is It Worth Looking for Mycobacteria?

The freshwater environment is suitable for nontuberculous mycobacteria (NTMs) growth. Their high adaptability represents a considerable risk for sanitary water systems, which are a potential vector for NTMs transmission. This study investigated the occurrence of NTMs, such as Mycobacterium saskatchewanense, in hospital water systems to support the surveillance and control of potentially pathogenic NTMs. We analyzed 722 ultrapure dialysis fluid samples from Emilia Romagna Dialysis Services. Among these, 35 samples were found to be positive for M. saskatchewanense. The strains were characterized using whole-genome sequencing (WGS) and variability analysis was carried out along the whole M. saskatchewanense genome. This investigation revealed the exclusive presence of M. saskatchewanense in these dialysis machines, with low genetic variability among all strains (with a low number of different alleles: <15). The strong similarity among the strain groups was also confirmed in the WGS-based ML tree, with very few significant nodes, and no clusters were identified. This research highlights the necessity of implementing surveillance protocols and investigating any potential link to human infections, as well as stressing the urgency of enhancing surveillance and infection control measures.

Pseudo-outbreak of haemodiafiltration dialysis fluid contamination: results of a detailed epidemiologic investigation

Compliance with dialysis fluid ultrapurity standards is a paramount for online modalities. More than 200 dialysis fluid samples have been analyzed monthly for years in our two dialysis units, with compliant microbiological results until mid-2020. In mid-2020, an unusual occurrence (30%) of contaminated dialysis fluids in dialysis units led us to investigate to determine the source. Microbiological methods for aquaphilic bacteria culturing and endotoxin detection in dialysis fluids were routinely performed on a monthly basis for all dialysis machines. As the contamination appeared randomly and almost simultaneously in our two units without any routine change or febrile syndrome, we searched for a common cause. Supplier's sampling kits as well as microbiological laboratory procedures were scrupulously investigated. 21 out of 30 sampling bags filled with sterile water brought back numerous fungi and bacteria. Laboratory's investigation, through the negative control tests performed routinely, exonerated the lab. All batches of bags analyzed later showed variable levels of contamination according to their transport/storage mode or date of manufacturing. Analyses performed by the supplier - methods complying with the medical device's standards but different from those recommended for dialysis fluids purity - remained negative. Our investigation revealed that the contamination of our sampling kits came presumably from the manufacturer's supplying chain. Such false-positive results findings, created serious safety issues and disturbed clinical activities since positive machines were quarantined. Furthermore, it raised a serious concern about manufacturing, microbiological checking and shipping methods for the medical device industry that deserve further attention.

Isolation and identification of potentially pathogenic free-living amoeba in dialysis fluid samples of hydraulic systems in hemodialysis units.

Free-living amoeba (FLA), including Acanthamoeba, Naegleria, Balamuthia and Vermamoeba, have been isolated from water, sand, soil, dust and air. Numerous studies considered that FLA are a significant cause of neurological and ocular complications in high-risk groups, including immunocompromised individuals. The present study aimed to identify morphological and molecular characteristics of FLA isolates in dialysis fluid samples of hydraulic systems in hemodialysis units in Iran. A total of 328 dialysis fluid samples were collected from 16 dialysis machines, including 164 samples before hemodialysis sessions (after cleaning) and 164 samples after hemodialysis sessions (before cleaning). Filtration and cultivation were performed on non-nutrient agar medium. Also, PCR and sequencing were applied by using the genus-specific primers along with a common primer set on positive samples. Both morphology and molecular investigations showed that 22.5% (74/328) of dialysis fluid samples were positive for FLA. There was a positive relationship between the high frequency of FLA after hemodialysis sessions (before cleaning) compared with before hemodialysis sessions (after cleaning) (OR=2.86; 95% CI 1.5 to 5.45). Considering the PCR assay, 16.46% (54/328) samples were identified as Acanthamoeba spp. (belonging to T3 and T4 genotypes), 5.18% (17/328) as Vermamoeba vermiformis and 0.91% (3/328) as Vahlkampfiidae family (Naegleria australiensis, Naegleria pagei and Allovahlkampfia). The present results support a need to improve filtration and purification methods for dialysis fluid of hydraulic systems in hemodialysis units. They also highlight the relevance of periodic screenings for FLA-related diseases in hemodialysis patients.

Comparison of techniques for culture of dialysis water and fluid.

Microbiological culture of dialysis water and fluid is a routine safety measure. In the United States (U.S.), laboratories perform these cultures on trypticase soy agar at 35-37°C for 48 h (TSA-48h), not on the tryptone glucose extract agar or Reasoner's 2A agar at 17-23°C for 7 days (TGEA-7d and R2A-7d, respectively) recommended by international standards. We compared culture methods to identify samples exceeding the accepted action level of 50 CFU/mL. Dialysis water and fluid samples collected from 41 U.S. dialysis programs between 2011 and 2014 were cultured at two U.S. laboratories. Each sample was cultured using (1) either TGEA-7d or R2A-7d and (2) TSA-48h. We compared proportions exceeding the action level by different methods and test characteristics of TSA-48h to those of TGEA-7d and R2A-7d. The proportion of water samples yielding colony counts ≥50 CFU/mL by TGEA-7d was significantly different from the proportion by TSA-48h (P = 0.001; difference in proportion 4.3% [95%CI 1.3-7.3%]). The proportions of dialysis fluid samples ≥50 CFU/mL by TGEA-7d and TSA-48h were not significantly different; there were no significant differences for comparisons of R2A-7d to TSA-48h. In dialysis fluid, TSA-48h was comparable to TGEA-7d and R2A-7d in identifying samples as having bacterial counts ≥50 CFU/mL. In dialysis water, TSA-48h was comparable to R2A-7d in identifying samples ≥50 CFU/mL, but TGEA-7d did yield significantly more results above 50 CFU/mL. Nonetheless, the negative predictive value of a TSA-48h result of <50 CFU/mL in dialysis water exceeded 95%.

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

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

Continuous Assessment of Intrahepatic Metabolism by Microdialysis During and After Portal Triad Clamping

Ischemia-reperfusion injury is a major concern with portal triad clamping (PTC) in liver surgery. Microdialysis allows continuous intraoperative monitoring of tissue metabolism in the liver. Our aim was to evaluate the feasibility of microdialysis as a tool to assess the intrahepatic metabolic effects of PTC in patients undergoing liver resection. Eleven patients who underwent liver resection were subjected to intrahepatic microdialysis. Dialysis fluid samples were collected before, during, and after a 20-min period of PTC. Glucose, lactate, pyruvate (markers of ischemia), and glycerol (marker of cell membrane damage) were analyzed and the lactate/pyruvate ratio was calculated. During PTC, intrahepatic glucose, lactate, and glycerol increased from 9.1±2.2 to 14.5±2.4 mM, from 2.2±0.3 to 5.8±0.5 mM, and from 63±14 to 142±28 μM, respectively. Pyruvate was unchanged, resulting in an increased lactate/pyruvate ratio (from 39±10 to 104±32). During initial reperfusion, glucose further increased to 16.4±2.9 mM. Pyruvate increased after reperfusion (from 93±18 to 138±23 μM), while lactate was stable, resulting in a normalized lactate/pyruvate ratio. Glycerol continued to increase during initial reperfusion. PTC was associated with considerable intrahepatic metabolic alterations with anaerobic metabolism, increased glycogenolysis, and cellular membrane damage resulting in increased levels of glucose, lactate, glycerol, and lactate/pyruvate ratio. Microdialysis is easy to use and allows continuous monitoring of intrahepatic metabolism during liver surgery.

A novel bio-assay increases the detection yield of microbiological impurity of dialysis fluid, in comparison to the LAL-test

Biological purity of dialysis water is considered as one of the primary conditions to deliver optimal haemodialysis. The present study explores the added value of a novel cytokine (IL-1ss) induction assay, using a monocytic THP-1 cell line, compared to the classical detection methods for microbial dialysis fluid contaminants. In contrast to the Limulus Amebocyte Lysate (LAL)-test, which only detects intact lipopolysaccharide (LPS), the THP-1 assay was also sensitive to peptidoglycan, short bacterial DNA fragments and LPS fragments <5 kD. The purity of 269 dialysis fluid samples was tested by the THP-1 assay and compared to the LAL-test. Two hundred and sixty samples complied with the definition of 'pure' dialysis fluid as laid down in the European Pharmacopeia (European Best Practice Guidelines for Hemodialysis. Section IV. Dialysis fluid purity. Nephrol Dial Transplant 2002; 17: 45-62) but 27 of these so-called pure dialysates (10.3%) provoked a pro-inflammatory response in the THP-1 assay. Furthermore, among the 230 samples that complied the definition of an ultrapure dialysis fluid, 21 samples (9.1%) were pro-inflammatory. These data illustrate that this novel bio-assay detects microbiological entities with an inflammatory potential that cannot be found by the classical LAL screening method. Adding this novel THP-1 assay to the classical methods will be helpful in the prevention of biofilm formation in the delivery system and should have relevance by more accurate detection of dialysate contamination, hence decreasing micro-inflammation in the haemodialysis patient.

Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining and microcolony methods

Bacterial contamination of dialysis fluids can cause inflammation and pyrogenic reactions in patients. Bacteria can grow more rapidly in dialysis fluids than in the high quality water used for their preparation, because dialysis fluids contain nutrients and electrolytes. Rapid determination of the microbiological quality of dialysis fluids is important, as it may aid in the reduction of instances of endotoxaemia. Microbiological contamination in dialysis systems should be detected as quickly as possible in order to provide dialysis fluid to patients with safety. Microbial contamination in dialysis fluids is usually assessed by culture-dependent methods such as plate counting. Arvanitidou et al. [1] examined dialysis fluids collected from 85 haemodialysis centres in Greece and found a total of 3500 5000 colony-forming units (CFUs)/ml of heterotrophic bacteria in 40% of measured samples [1]. Oie et al. [2] reported that dialysis fluids are more frequently contaminated than the water, acid or bicarbonate concentrates used for dialysis fluid preparation, and that the bacterial count in 42.5% of 40 dialysis fluid samples was >2000CFU/ml. These data indicate that the microbial contamination levels in a considerable proportion of dialysis fluid samples are relatively high. However, culture-dependent methods often underestimate bacterial numbers, because many bacterial species such as mycobacteria cannot be cultivated by conventional means [3], thus potentially placing the patients at a higher risk of inflammation and pyrogenic reactions. In addition, plate counting can require up to several days to detect bacteria. Therefore, microbiological qualities of dialysis fluids are often evaluated by measuring the endotoxin level. The endotoxin assays are simple and require a few hours to obtain results. However, endotoxin levels are often not correlated with numbers of CFU, and high numbers of colonyforming cells are sometimes detected in dialysis fluids with significantly low endotoxin level [4]. Simple, rapid and accurate methods of enumeration without longtime (e.g. 7 days) culture are essential, in order to maintain microbiological quality of dialysis fluids. Fluorescent staining is widely applied to detect bacteria rapidly [5]. Fluorescent dyes stain the nucleic acid and/or protein within the bacterial cells, and these cells can be counted by fluorescent microscopy, laser scanning cytometry and flow cytometry. Fluorescent vital staining such as 40,6-diamidino-2-phenylindole (DAPI) – carboxyfluorescein diacetate (CFDA) double staining can simultaneously determine the total bacterial count and the number of physiologically active bacteria (bacteria with enzymatic activity), not only in environmental samples (river water, pond water, ground water) but also in medical and pharmaceutical samples (pharmaceutical water [6] and herbal medicines [7]). By this technique, numbers of total bacteria (both active and inactive cells) are determined by DAPI staining, and physiologically active cells are selectively enumerated with CFDA staining, without the need for culture. DAPI is used for total direct counting of microbial cells in aquatic environments, because of its high stainability [5]. CFDA is a popular, ultra-sensitive, fluorescent substrate for estimating esterase activity in live micro-organisms (bacteria, yeast and fungi) [5]. Activity of cytoplasmic esterase of micro-organisms is an indicator of their physiological activity. Not only cells with growth potential but also dormant or injured cells can be stained with CFDA. Many bacteria in water with low nutrient concentrations cannot form large colonies which are Correspondence and offprint requests to: Masao Nasu, Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Email: nasu@phs.osaka-u.ac.jp Nephrol Dial Transplant (2007) 22: 612–616

Filtration fluid for hemodialysis treatment.

Bacterial contamination of dialysis fluid has long been recognized as a problem in hemodialyis. Cytokines released as a consequence of contaminated dialysis fluid are believed to be responsible for many acute and chronic side effects in patients undergoing renal replacement therapy. For several years now, attempts have been made to eliminate pyrogenic substances and ensure a sterile and endotoxin-free dialysis fluid. A recent dialysis fluid filter known as DIASAFE, containing a membrane based on Polysulfone (Fresenius), was tested for a period of 1,000 hours (approx. 14 weeks). Dialysis fluid samples were collected once weekly before and behind the filter and cultivated for detection of microorganisms and endotoxins. Additionally, starting after the fourth week of the study, serum samples were collected weekly and the beta2-microglobulin concentration was determined. The filter reduced microorganisms at a rate of at least 10(5) and in the majority of cases (86% of samples) by more than 106. Under clinical conditions the stability and microbiological functionality of the filters could be demonstrated for more than 1,000 hours and 150 disinfecting cycles. In four cases of endotoxin burden (> 0.5 IU/ml) in the dialysis fluid in front of the filter the concentration behind the filter was lower than 0.1 IU/ml, indicating effective reduction of endotoxins. A tendency to a reduction of beta2-microglobulin in serum from 32.5+/-3.9 mg/L to 21.5+/-5.3 mg/L was observed. These results indicate that the dialysis fluid filter used was effective, dramatically reducing the bacterial contaminants in dialysis fluid, thus protecting patients from the potentially harmful acute and long-term life-threatening consequences of contaminated dialysis fluid.

QUALITY CONTROL OF DIALYSIS FLUIDS BY MICELLAR COLORIMETRY AND MULTIVARIATE CALIBRATION

A colorimetric method for the determination of zinc, copper and manganese as a quality control criterion in dialysis fluids is discussed. The spectrophotometric reagent was an aqueous solution of 1-(2-pyridylazo)-2-naphthol (PAN) in Triton (5% v/v) in order to obtain soluble micelles of the reagent and its chelates to perform measurements in aqueous phase. Multivariate calibration by the classical least square (CLS) method was adequate to assay the analytes and to verify the presence of other contaminants. Solutions of Zn, Cu and Mn ions in borate buffer (pH 9.2) were used to obtain the spectroscopic data for the calibrations. The modelling was investigated using “one analyte” standard solutions (Zn, Cu or Mn) and also mixtures of standard solutions (Zn, Cu and Mn). The results were compared in terms of the calculated RMSEP (Root Mean Square Error of Prediction) values. Recoveries about 100% were calculated using the methodology in synthetic samples of dialysis fluids and the interference of Ca, Mg, Fe, Cd, Ni and Co ions was discussed. Concentrations of the analytes in the range of 6–500 μg L−1 were determined in real dialysis fluid samples using “one analyte” standard solutions for the calibrations and the results were compared with atomic absorption spectrometry. Calibrations using PCR (Principal Component Regression) and PLS (Partial Least Square) methods showed similar results as those obtained by CLS method.

Determination of trace elements in a large series of spent peritoneal dialysis fluids by atomic absorption spectrometry

An analytical procedure is reported for the determination of six elements in a large series of spent dialysis fluid samples. Determinations of aluminium, chromium, copper, manganese and iron were made by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman background correction, while zinc was analysed by flame atomic absorption spectrometry (FAAS). Because of the complex matrix with high salt content and a high content of proteins, the measurement parameters were optimised for each particular element determined by ETAAS. The samples were collected in polyethylene eppendorf cups and stored in a freezer at −20°C. When the elements were determined by ETAAS the standard addition method was applied in the calibration procedure. The sample (10 μl) was injected into a cuvette and careful drying and long ashing of samples at temperatures between 850 and 1000°C performed. Triton X-100 was added before each determination to reduce the matrix effects of the proteins. Zinc was determined by FAAS in an air–acetylene flame under the usual recommended procedure, calibrating with aqueous standards. The limits of detection (3 σ basis) were 1.0 μg l −1 for aluminium, 0.20 μg l −1 for chromium, 0.40 μg l −1 for copper, 0.20 μg l −1 for manganese, 0.50 μg l −1 for iron and 5.0 μg l −1 for zinc. The reproducibility of the measurements for aluminium, copper, iron and zinc was better than ±3.0%. It was worse for manganese and chromium (±6.0 and ±12.0%, respectively), since these two elements were present in very low concentrations in all the samples analysed.

Prevention of aluminium exposure through dialysis fluids. Analysis of changes in the last 8 years.

Despite extensive measures to control aluminium exposure, chronic and acute episodes of aluminium intoxication still occur. The objective of this study was to analyse the changes in the aluminium content of dialysis fluid and the effect on serum aluminium in different dialysis centres in Spain in the last 8 years. For this purpose, the aluminium content in dialysis fluid and serum samples (N=5609) from 17 dialysis centres was analysed for >8 years (from the last quarter of 1988 to 1996). In that period of time, the percentage of dialysis fluid samples with acceptable concentrations of aluminium (<2 microg/l) increased from 0% in 1988 to 80% in 1996. The percentage of dialysis fluid samples with high aluminium levels (>6 microg/l) ranged between 37.5% in 1988 and 2.3% in 1996. The improvement in the quality of the dialysis fluid resulted in lower values of serum aluminium. The percentage of serum samples with low aluminium (<20 microg/l) increased from 16.5% in 1988 to 54.2% in 1966. The mean serum aluminium correlated with the mean dialysis fluid aluminium (r=0.55, P<0.001). A higher correlation was found when the aluminium in dialysis fluid ranged between 4 and 10 microg/l (r=0.802, P<0.001), and no correlation was found when the aluminium in dialysis fluid was <4 microg/l. Even taking into account that the dialysis fluid is not the only source of aluminium for dialysis patients, our study clearly demonstrated a close relationship with the serum aluminium content. Therefore, we must emphasize the necessity for controlling the aluminium content in dialysis fluid more often than is done at present.

Occurrence of endotoxin in dialysis fluid from 39 dialysis units

Endotoxin exposure during haemodialysis may cause acute and chronic adverse reactions. In order to estimate the risk to the patient, samples of dialysis fluid from 39 of the 45 dialysis units in Sweden were analysed by 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 1 −1), while 18% of units had high levels (mean concentration >100 ng 1 −1).

Influence of Storage Conditions on Aluminum Concentrations in Serum, Dialysis Fluid, Urine, and Tap Water

The influence of storage temperature, vessel type, and treatment on alterations of aluminum (Al) concentrations in serum, urine, and dialysis fluid samples was studied at three different concentrations for each sample over an 18-month period. Furthermore, the influence of acidification on Al levels in tap water, urine, and dialysis fluid samples was studied over a four-month period. Al was measured by atomic absorption spectrometry. Sample storage in glass vessels was unsuitable, whereas only minor alterations of Al levels were observed with storage in polypropylene tubes, polystyrene tubes, and Monovettes. By using appropriate plastic containers, acid washing of the vessels showed no improvement. Frozen storage was superior compared with 4 degrees C, whereas storage at -80 degrees C offered no advantage compared with storage at -20 degrees C. Acidification of tap water samples was necessary to stabilize Al levels during storage. No striking effect of acidification on Al levels in urine and dialysis fluid samples was found. It is concluded that longterm storage of serum, urine, tap water, and dialysis fluid samples is possible if appropriate conditions are used.

Liquid-chromatographic assay of cefamandole in serum, urine, and dialysis fluid.

We describe a "high-performance" liquid-chromatographic assay for quantifying cefamandole in biological fluids from patients with renal impairment. Serum samples are deproteinized with acetonitrile, then extracted with dichloromethane; dialysis-fluid samples are injected directly; urine samples are diluted appropriately before injection onto the reversed-phase column. The mobile phase is a methanol/aqueous solution (31/69 by vol) containing 500 microL of phosphoric acid, 20 mmol of sodium sulfate, and 200 microL of triethylamine per liter, the mixture being adjusted to pH 6.0 with NaOH. Retention time for cefamandole is 12 min. Its peak is well resolved in highly contaminated samples from renally impaired subjects. The assay's selectivity, reproducibility (within-day and between-day CVs less than 8% in all three sample fluids), and sensitivity--0.5 mg/L in serum, 1.0 mg/L in dialysis fluid, and 5.0 mg/L in urine--make it applicable to pharmacokinetic studies.