Materials and Methods

Study Design

The study was divided into two phases.

Phase 1 was used for the detection of prevalence and the level of inflammation in the entire dialytic population of the Vicenza hemodialysis center. Blood samples were collected from 81 pa tients under sterile conditions immediately after needle insertion but before any intravenous fluid was given (mid-week dialysis session) to measure the inflammatory parameters and make microbiological analyses (standard and molecular).

Phase 2 was used for the molecular evaluation of the presence of bacterial DNA in patients and hemodialyzers. 38 patients (of the original 81) without evident clinical infection or clear causes of inflammation were selected for this study. Whole blood and dialysate were collected during treatment. Blood was collected just after needle insertion and spent dialysate was collected every hour (4 samples of 50 ml). Samples from the blood and dialysate compartments of the dialyzers were collected following treatment and after filter washing, also collecting the last washing solution for control. Patients were excluded if they had apparently active infection or antibiotic administration within 2 weeks, and/or if other sources of inflammation such as periodontal disease, malignancy, autoimmune disease, trauma, infarction, etc., were present.

Controls

In phase 1 the whole blood of 20 healthy blood donors was collected for the inflammation study.

Microbiological study controls in phase 1 were internal controls for amplification (DNA from Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, P. aeruginosa ATCC 35218, Candida albicans ATCC 90028); whole blood of 20 healthy blood donors, and dialysis ultrapure water collected from different points of the treatment plant.

In phase 2 the microbiological study control was performed through simulated use of sterile hemodialyzers and water monitoring. Five unused hemodialyzers were treated in the same way as the patients' hemodialyzers after treatment, and microbiological monitoring of ultrapure water was collected from 6 different points of the water treatment plant.

Inflammatory Markers

The inflammatory markers evaluated have been divided into three categories.

(1) Inflammation Markers Modified in Response to Inflammation. High-sensitivity C-reactive protein (hsCRP): CRP (^mol/l) expresses an enhanced hepatic synthesis of proteins activated by conditions of chronic or repeated immune challenge. Interleukin 6 (IL-6) was measured as ng/l. The cells of the immune system are influenced by the toxic effect of uremia and by different dialytic procedures. Patients on renal replacement therapy are at high risk of infectious complication. It appears that in uremia B-cell function is normal, but there is a defect in T-cell function. During the interdialytic interval, cytokine production from monocytes is normal, even though these cells release large amounts of proin-flammatory cytokines such as IL-6. Small bacterial DNA fragments [17] and contaminants such as lipopolysaccharide are able to induce IL-6 in human mononuclear cells. Albumin (g/dl) was measured in patients with inflammation, infection or injury, and the catabolism and transendothelial transport of albumin may be increased while its synthesis is decreased.

(2) Markers of Immune Disregulation. The following parameters were evaluated. Percent monocytes HLA-DR+ was determined by flow cytometric analysis. HLA-DR expression, measured by flow cytometric analysis as medium fluorescence intensity (MFI) DR, may be an important parameter to evaluate the function of immunocompetent cells. A low expression can lead to severe immunodeficiency and has been associated with an increased risk of infection after surgery or trauma. Apoptosis, measured as a percentage of cells after incubation of patient plasma on U937 cells for 96 h, can be correlated to HLA-DR since it is related to defective immunity. Hemodialysis patients show a higher rate of apoptosis compared to healthy people, which is possibly related to retained uremic toxins. U937 in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/mlpenicillin, 100 mg/ml streptomycin were kept in a controlled atmosphere incubator (5% CO2) at 37°C. 106 U937 cells were incubated with patient and donor plasma plus 0.5 ml RPMI solution. Apoptosis was assessed by fluorescence microscopy after 96 h of incubation [18, 19].

(3) Causes of Inflammation (Oxidative Stress). CKD patients undergoing hemodialysis present an imbalance in oxidative equilibrium, characterized by a reduction in oxygen radical scavenger activity and an enhanced production of reactive oxygen species leading to an acute/chronic inflammatory response. As oxidative stress parameters we evaluated advanced oxidation protein products (AOPPs), glutathione (GSH) and reactive carbonyl compounds (RCOs). AOPP is a marker of oxidative stress and is formed by myeloperoxidases and chlorinated oxidants generated by neutrophils. Determination of AOPP was performed by spectrophotometry (absorbance reading 340 nm) and the concentration is expressed as nmol/l of chloramine T equivalents. GSH, measured as ^mol/106 cell, is the major determinant of the redox status in mammalian cells. It maintains intracellular redox equilibrium and regulates cellular defenses augmented by oxidative stress. Patients with endothelium dysfunction have lower GSH

[20]. RCOs are derived by carbohydrates, lipids and amino acids which become the precursors of RCOs in hemodialysis patients. Determination of RCOs was performed by spectrophotometry (absorbance reading 370 nm) using dinitrophenylhydrazine binding and the concentration is expressed as nmol/l of proteins

DNA Extraction from Whole Blood

DNA from 200 ^l of heparin-treated whole blood was extracted using a QIAamp DNA mini kit (Qiagen) following the manufacturer's instructions. DNA was digested with proteinase K in an appropriate buffer for 2 h at 56°C to allow optimal cell lysis and binding of the DNA to the QIAamp membrane. DNA was then adsorbed onto the QIAamp silica gel membrane during brief cen-trifugation. Salt and pH conditions ensured that proteins and other contaminants were not retained on the membrane. DNA bound to the membrane was washed twice in two brief centrifugations using two different buffers which significantly improves the purity of the eluted DNA without affecting DNA binding. Purified DNA was eluted with an elution buffer in a concentrated form and was then suitable for direct PCR use.

DNA Extraction from Dialysate

200 ml of dialysate collected during dialysis was centrifuged at 2,000 rpm for 10 min in order to pellet bacterial cells. The pellet thus obtained was digested overnight in 10 mM Tris-HCl (pH 8.3) and mM KCl 50 with proteinase K to a final concentration of 0.5 ^g/^l and Nonidet P-40 at 55°C. The mixture was then boiled for 10 min and centrifuged to remove debris. The supernatant was used as template for amplifications.

DNA Extraction from Dialyzers

25 ml of a mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, proteinase K to a final concentration of 0.5 ^g/^l and Nonidet P-40 was injected separately into the blood and the di-alysate compartments of the filters. The filters were then incubated at 42°C overnight to allow complete digestion of biofilm if present. Then, in order to avoid the cross-contamination between the solutions in the two compartments, we separately removed the two solutions by gentle drawing them from the arterial and dialysate ports in two sterile tubes. The solutions were boiled for 10 min and centrifuged to remove debris. The supernatant was used as direct template for amplifications. All processes were done under sterile techniques.

DNA Isolation and Extraction from Ultrapure Water

In order to evaluate microbiological quality, 200 ml ultrapure water for dialysis were collected at different points in the treatment water plant (formula 10, loop 1, loop 2, Fresenius, AK 2000 and integra) and filtered in a 0.2-^m Millipore membrane. The membrane was then treated to extract DNA, if present, by means of 10 mM Tris-HCl (pH 8.3) and 50 mM KCl with proteinase K to a final concentration of 0.5 ^g/^l and Nonidet P-40 at 55°Cover-night. The mixture was then boiled and ready for amplification.

16S rRNA Amplification. It was shown that the phylogenetic relationships of bacteria and, indeed, all life-forms could be determined by comparing a stable part of genetic code [20, 21]. The DNA part now most commonly used for taxonomic purposes for bacteria is the 16S rRNA gene [13]. In this study, the primers used for amplification of 16S rRNA were 355F (5'-CCTACGGGAG-GCAGCAG-3') and 910R (5'-CCCGTCAATTCCTTTGAGTT-3'). 200-1,000 ng of template DNA were used for amplification in a 50-^l reaction mixture with a final concentration of 67 mM Tris HCl (pH 8.8), 16 mM (NH4)2SO4, 200 ^M dNTPs, 3,5 mM MgCl2, 25 pmol of each primer and 1 U Taq polymerase (GoTaq DNA polymerase, Promega, Madison, Wisc., USA). The temperature scheme used for the amplification was: 95°C for 5 min then 35 cycles of 95°C for 45 min, 53°C for 45 min and 72°C for 45 min and a final extension step of 7 min at 72 °C. The amplification products were visualized in 3% Nu:Sieve 3:1 Agarose (Cambrex Bio Science, Rockland, Me., USA) with 5% gel star staining (Cam-brex) using standard techniques. All samples were tested at least twice before reporting. To avoid risk of contamination, tissue preparation, PCR amplification and electrophoresis were performed in different rooms. In each assay negative and positive controls were run. The negative control contained all the PCR reagents and sterile bi-distilled water.

Precautions taken to avoid laboratory contamination included: the use of different areas for pre-PCR preparation and sample preparation and managing (post-PCR area); the use of face masks, gloves, caps and glasses, and the use of barrier tips and different pipettes when handling reagents and specimens in the pre- and post-PCR areas. Native Taq polymerase from Thermophilus aquaticus, instead of recombinant, was used to avoid the presence of contaminant DNA from commonly found bacteria. A negative and a positive control were included in each run. All procedures were performed after at least 12 h of UV sterilization of the workplace, and amplification of the p-globin gene was performed to evaluate sample degradation or the presence of inhibitors.

Amplification Products Excision from Gel and DNA

Sequencing

After electrophoresis, the amplification products, if present, were excised from gel and purified with Wizard SV Gel and PCR Clean-up System (Promega, cat. No. A9282) following the manufacturer's instructions. The products then underwent sequencing reaction on GeneAmp 9700 (PE Biosystems). The ABI Prism Big-Dye Terminator v1.1 cycle sequencing kit (Applied BioSystems, Foster City, Calif., USA) was used for the sequencing reaction; the primer used was the reverse (910 R) and the final reaction volume was 20 ^l. The thermal cycling conditions were 25 cycles of 10 s at 96°C, 5 s at 50°C and 4 min at 60°C. The reaction products were purified with Centri-Sep Columns (Princeton Separation) to remove exceeding DyeDeoxy™ terminators before automated sequencing on ABI PRIMS 310 genetic analyzer (Applied BioSystems). The sequences obtained were examined on the web site http://www.ncbi.nlm.nih.gov/blast.

Hemocultures

A minimum of 10 ml of blood was obtained and immediately inoculated into BacT/Alert Fanh aerobic and anaerobic bottles (BioMerieux, Marcy I'Etoile, France), and the bottles were incubated for <7 days. The bottles were then processed in a BacT/ Alerth 3D automated blood culture system (BioMerieux). Blood cultures were performed at the time of filter and ultrafiltrate collections and at the beginning of the hemodialysis session.

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