Materials and Methods

Kidney Function Restoration Program

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Study Subjects and DNA Extraction

Nine critically ill patients with acute renal failure (ARF) requiring CRRT were included in the prospective observational cohort study. For all subjects a diagnosis of 'severe sepsis' was made according to the criteria of Bone et al. [8] including (1) at least 2 systemic inflammatory response syndrome (SIRS) criteria; (2) confirmed or suspected infection, and (3) multiple organ dysfunction syndrome. The study protocol was approved by the hospital ethics committee.

All septic patients were treated following the guidelines for severe sepsis and septic shock [ 9]. Broad-spectrum antibiotics were given to all patients. The clinical characteristics and antibiotic prescriptions are given in table 1 . Blood cultures, and blood samples/filters/UF collection for bacterial DNA detection were performed when severe sepsis was diagnosed.

For each subject at least 3 samples for blood culture were obtained from different sites by peripheral venipuncture. In case of suspected catheter-related sepsis, 1-2 blood samples for culture were drawn from the catheter. 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) [10].

After blood samples had been drawn, DNA from 0.2 ml of heparin-treated whole blood was extracted by means of a commercial DNA extraction kit (QiAmp DNA Minikit, Quiagen Science, Valencia, Calif., USA) according to the manufacturer's instructions. Briefly, DNA was digested with proteinase K and an appropriate buffer for 2 h at 56° C to allow optimal lysis and binding of the DNA to the QiAamp membrane. DNA was adsorbed onto the QiAamp silica gel membrane by centrifugation. Salt and pH conditions ensured that proteins and other contaminants were not retained on the membrane. In order to wash DNA that was adsorbed into the membrane, two different buffers were utilized which significantly improved the purity of the eluted DNA without affecting DNA binding. Purified DNA was then eluted with a buffer in a concentrated form and was suitable for direct polymerase chain reaction (PCR) use.

At the end of a 24-hour CRRT session 200 ml of UF was collected and centrifuged at 2,000 rpm for 10 min in order to pellet bacterial cells if present. After centrifugation, the pellets obtained were digested overnight in a lysis buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and proteinase K to a final concentration of 0.5 ^g/^l and Nonidet P-40 (Roche Applied Science, Mannheim, Germany) at 55°C. The mixture was then boiled for 10 min and centrifuged to remove debris. The supernatant was used as a template for amplifications. Direct precipitation of DNA with sodium acetate 3M and cold absolute ethanol (-20°C) and subsequent water dilution were also performed to evaluate the presence of DNA in solution. Both samples were analyzed for the presence of bacterial DNA.

Filters. 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 into the blood compartment and the dialysate compartment of the filters. The filters were then incubated at 37° C overnight to allow complete digestion of biofilm if presented. Then, in order to avoid cross-contamination between the solutions in the two compartments, we separately removed the two solutions by gentle drawing from the arterial and dialysate ports and collected the fluid in two sterile tubes. The solutions were boiled for 10 min and centrifuged to remove debris. The supernatant was used as a direct template for amplifications. All processes were done under sterile techniques.

rRNA 16S PCR Amplification and DNA Sequencing

It was shown that the phylogenetic relationships of bacteria, and indeed all life-forms, can be determined by comparing a stable part of the genetic code [11, 12]. 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'-CCTACGGGAGGCAGCAG-3') and 910R (5'-CCCGTCAATTCCTTTGAGTT-3'): these primers detect a 540-bp region of the 16S rRNA gene. Template DNA was used for amplification in a 50-^l reaction mixture at 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 (Eurobio, Courtaboeuf, France). The temperature scheme used for the amplification was: preheating at 95°C for 5 min followed by 35 cycles of 95°C for 45 s, 53°C for 45 s, 72°C for 45 s, and a final extension step of 72°C for 7 min. The amplification products were run through 3% Nu:Sieve 3: 1 Agarose (Cambrex BioScience,

Table 1. Clinical characteristics of the patients studied

Subjects

Table 1. Clinical characteristics of the patients studied

Subjects

1

2

3A

3B

4

Age, years/gender

69/M

60/M

51/F

51/F

52/M

Diagnosis on ICU

perforated ileum,

catheter-related sepsis

catheter-related sepsis

after catheter-related

acalculous cholecystitis,

admission

peritonitis

sepsis (3 weeks)

sepsis

Comorbidities

ESRD on CAPD

ESRD, DM, HT

ESRD, DM, HT

ESRD, DM, HT

coronary artery disease,

CHF

Antibiotics used

tazocin,

oxacillin, gentamycin

tazocin

nil

tazocin, ceftazidime,

metronidazole,

amikacin, fluconazole

fluonazole

Type of RRT

CVVH

IHD

IHD

IHD

CVVH

Hemofilter

AV 1000 S (Fresenius)

F10 HPS (Fresenius)

F10 HPS (Fresenius)

F10 HPS (Fresenius)

Diafilter D30 (Minntec)

Membrane type

polysulfone

polysulfone

polysulfone

polysulfone

polysulfone

Surface area, m2

1.8

2.4

2.4

2.4

0.7

Kuf, ml/h/mm Hg/m2

52

18

18

18

Subjects

Age, years/gender Diagnosis on ICU

admission Comorbidities

Antibiotics used

Type of RRT Hemofilter

Membrane type

Surface area, m2 Kuf, ml/h/mm Hg/m2

56/F

bowel ischemia, sepsis

CA colon, after colectomy

45/M

broncopneumonia, septic shock leukemia, leukopenia, thrombocytopenia amikacin, astreonam, linezolid, tazocin, metronidazole CVVH Diafilter D30 (Minntec) polysulfone

clarithromycin CVVH Diafilter D30 (Minntec) polysulfone

37/M

sepsis? acute rejection?

kidney transplant, graft failure (renal vein of transplant kidney and iliac vein thrombosis)

79/M

colon cancer, septic shock after colectomy DM

ceftriaxone IHD

NC 2085 (Bellco)

ciprofloxacin, metronidazole, tazocin CVVH

AV 600 S (Fresenius)

synthetically modified polysulfone cellulose

46/F

sepsis (unknown source)

kidney transplant, acute graft rejection levoflocaxin, tazocin, fluconazone

CVVH

Diafilter D30 (Minntec)

polysulfone

ESRD = End-stage renal disease; DM = diabetes mellitus; HT = hypertension; CHF = congestive heart failure; CA = cancer; RRT = renal replacement therapy; CVVH = continuous veno-venous hemofiltration; IHD = intermittent hemodialysis.

Rockland, Me., USA) with 5% gel star staining (Cambrex Bio Science) using standard techniques. All samples were tested at least twice before reporting. In order to avoid risk of contamination, tissue preparation, PCR amplification and electrophoresis were performed in different rooms. In each assay a negative and a positive control were run. The negative control contained all the PCR reagents and sterile bi-distilled water.

After electrophoresis of the amplification products, if present, the PCR products were excised from gel and purified with Wizard SV Gel and PCR Clean-up System (Promega Corp., Madison, Wisc., USA) according to the manufacturer's instructions. The products then underwent sequencing reactions on GeneAmp 9700 (PE Applied Biosystems, Foster City, Calif., USA). A BigDye Terminator vl.1 Cycle sequencing kit (Applied BioSystems) was utilized for the sequencing reaction. The primer used as the template was the reverse one (910R) and the final reaction volume was 20 ^l. The thermal cycling conditions were 25 cycles of 96°Cfor

10 s, then at 50 ° C for 5 s and at 60 ° C for 4 min. The reaction products were purified with Centri-Sep 8 Columns (Princeton Separation, Adelphia, N.J., USA) to remove unincorporated dye terminators, and the sequence was determined using capillary electro-phoresis (ABI PRISM 310 Genetic Analyzer, Applied BioSystems). The generated DNA sequences were then compared with a database library on the GenBank web site (http://www.ncbi.nlm.nih. gov/blast).

Prescription of Renal Replacement Therapy Techniques

Continuous veno-venous hemofiltration (CVVH) was started in each case when a 'failure' level was denoted using the RIFLE criteria. RIFLE is an acronym indicating: Risk of renal dysfunction; Injury to the kidney; Failure of kidney function; Loss of kidney function, and End-stage kidney disease [14], Thus, criteria were a threefold increase in the creatinine level with respect to baseline, or a creatinine concentration of >4 mg/dl, or oligo-

Table 2. Microbiologic results

Subjects

Table 2. Microbiologic results

Subjects

1

2

3A

3B

4

Blood culture (no. of sets)a

negative (3)

Staphylococcus aureaus (2/3)

Pseudomonas aeruginosa (3/3)

negative (3)

negative (5)

Other cultures (site)

P. aeruginosa (bile, abdominal drainage, BAL, tracheal aspiration)

Bacterial DNA detected b

Blood

negative

non-sequentiable

Pseudomonas spp.

negative

non-sequentiable

Filter

Blood compartment

negative

non-sequentiable

Pseudomonas spp.

non-sequentiable

non-sequentiable

Dialysate compartment

negative

uncultured bacteria

Pseudomonas spp.

uncultured proteobacterium

Pseudomonas spp.

Ultrafiltrate

negative

negative

negative

negative

Subjects

Blood cultures (no. of sets)a Other cultures (site)

Bacterial DNA detectedb

Blood

Filter

Blood compartment Dialysate compartment Ultrafiltrate negative (7) Streptococcus agalactia gr. B (tracheal aspiration)

negative non-sequentiable non-sequentiable negative negative (3) negative (tracheal aspiration)

negative non-sequentiable negative negative negative (3) negative (urine)

non-sequentiable non-sequentiable negative negative negative (3) negative (BAL)

Pseudomonas spp.

Pseudomonas spp. Pseudomonas spp. negative negative (4) negative (urine, BAL)

Candida albicans

Candida albicans Candida albicans negative

BAL = Bronchoalveolar lavage.

a Each set consists of one aerobic and one anaerobic bottle. b Bacterial DNA was detected by bacterial 16S rRNA gene sequence analysis.

anuria lasting for 24 h. 1.8 m2 polyethersulfone high-flux hemo-filters were utilized. A bicarbonate-based solution was used as replacement fluid. Blood access was established with 13-french double-lumen hemodialysis catheters. The blood flow rate was 150-180 ml/min and an ultrafiltration flow rate of 35 ml/kg/h was used in the pre-dilution mode. Prescription was performed with the help of a calculator recently described by our group [15]. In the absence of contraindications, low dose (250-500 U/h) pre-filter unfractionated heparin was infused to prevent filter clotting. According to hospital policy, we changed the hemofilter after 24 h of uninterrupted CVVH treatment. Two milliliters of blood and 200 ml of UF were withdrawn from the arterial and effluent ports, respectively, immediately before changing the filters. Then the hemofilters were cleaned with 200 ml of sterile normal saline and removed and the arterial, venous and dialysate ports were closed with sterile taps. All of these processes were done under sterile conditions. Blood was collected into refrigerated tubes containing solid heparin, and UF was collected into four 50-ml sterile tubes.

Statistical Analysis

The x2 test was performed in order to compare if molecular techniques differed in detecting or not detecting the presence of the bacteria with respect to standard blood culture. A p value of <0.05 was considered statistically significant.

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