Results

Phase 1

Inflammatory Markers

The inflammatory markers were evaluated in the whole population (81 patients) and in the same population divided into 2 subgroups: a group in which evident causes of inflammation could be clinically demonstrated (43 patients), and a group with inflammation but no evident causes for it (38 patients). The results are shown in table 1. Comparison of the 43 patients with known causes of inflammation with the 38 patients without causes of inflammation reveals a trend to an increase in IL-6 in the second group. Other parameters are not significant. Thus, the level of inflammatory parameters is not significantly different in the 2 groups, suggesting that a similar level of inflammation may be present. A further consideration is that, in general, the level of inflammatory markers is remarkably low compared to other populations.

Microbiological Parameters

The whole blood from all 81 patients was hemoculture negative, while 11 of 81 (13.58%) patients had positive bacterial DNA in the whole blood. After sequencing 8 had Pseudomonas spp. DNA while the last 2 had a strong signal, but no readable sequence. All controls (blood donors

Table 1. Inflammatory markers evaluated in the whole population and in the two subgroups 43 patients with evident causes of inflammation and 38 patients with inflammation but without evident causes

Population Inflammatory response (markers/cytokine) Immune disregulation Causes of inflammation (oxidative stress)

Table 1. Inflammatory markers evaluated in the whole population and in the two subgroups 43 patients with evident causes of inflammation and 38 patients with inflammation but without evident causes

Population Inflammatory response (markers/cytokine) Immune disregulation Causes of inflammation (oxidative stress)

HsCRP mg/dl

IL-6 pg/ml

albumin g/dl

AOPP |M

|xmol/106 cells

RCOs nmol/mg protein

apoptosis plasma on U937 cells at 96 h, %

HLA-DR+

%

Total (n = 81)

1.0681.21

17.56827.34

3.868 0.43

237.638136.67

5.0481.06

1.0080.56

45.1480.07

99.28 40.46

96.478 3.83

Evident causes of inflammation (n = 43)

1.11 ± 1.27

11.08811.44

3.808 0.46

217.058117.85

4.9381.08

0.998 0.28

46.778 0.07

91.63842.16

95.868 3.97

No evident causes of inflammation (n = 38)

0.99 81.16 n.s.

24.738 36.75 p = 0.034

3.938 0.40 n.s.

260.938153.51 n.s.

5.1781.03 n.s.

1.0080.76 n.s.

44.428 0.07 n.s.

107.76837.14 p = 0.073

97.1783.59 n.s.

Table 2. Correlation of molecular data obtained in whole blood and inflammatory markers in the whole population (81 patients)

Population

Inflammatory response (markers/cytokine) Immune dysregulation

HsCRP mg/dl

IL-6 Pg/ml albumin g/dl oxidative stress determined by AOPP, |xmol/l oxidative stress determined by GSH |imol/g Hb

Causes of inflammation (oxidative stress)

carbonyl stress nmol/mg protein apoptosis plasma on U937 cells at 96 h, %

MFI DR+

monocytes

HLA-DR+

22.41 16.78

257.55 234.5

75.79 102.87

and ultrapure water samples) had negative hemoculture and negative bacterial DNA. Internal controls for amplification were used to standardize the molecular protocol.

Inflammatory Markers vs. Molecular Data in All

Patients

Inflammatory markers were compared to the molecular data obtained from whole blood. The statistical correlation was not significant because of the limited number of patients, but a trend can be noticed for an increase in IL-6 and carbonyl stress (RCOs) and a reduction in MFI in bacterial DNA-positive patients when compared with bacterial DNA-negative patients. The results are summarized in table 2 .

Phase 2

Molecular Evaluation of Patients with No Evident

Causes of Inflammation

The molecular evaluation of the 38 patients (of the original 81) without clinical infection or clear causes of inflammation was performed in whole blood, spent di-alysate and hemodialyzers (blood and dialysate compartments). 34 (89.5%) patients were positive in one or more of the collected samples, while only 4 (10.5%) were com pletely negative. Four of 38 (10.5%) patients had bacterial DNA in whole blood collected after needle insertion prior to any fluid infusion: 2 had Pseudomonas spp., and 2 could not be sequenced. Seven of 38 (18.4%) patients had bacterial DNA in spent dialysate collected every hour during treatment: 1 could not be sequenced, and 6 had Pseudomonas spp. 19 of 38 (50%) patients had bacterial DNA in the blood compartment of the hemodialyzer: 10 could not be sequenced, and 9 had Pseudomonas spp. 23 of 38 (60.5%) patients were positive in the dialysate compartment: 4 could not be sequenced; 15 had Pseudomonas spp., and 4 had environmental infections such as Halomo-nas spp., Proteobacterium unc. Dividing patients for pos-itivity according to different compartments (blood side considering whole blood and blood compartment, and dialysate side considering spent dialysate and dialysate compartment), the results are as follows: none were only whole blood or spent dialysate positive; 7 patients were only blood compartment positive; 9 patients were only dialysate compartment positive; 2 were whole blood and blood compartment positive; 2 were dialysate compartment and spent dialysate positive, and 14 were positive in more than one sample (different compartments). The results of sequencing are reported in table 3.

Table 3. Molecular evaluation and sequencing in a population with no evident cause of inflammation divided for positivity in one or more different compartments

Positive collection points

Bacterial DNA-

Sequencing

positive patients

Whole blood only

0

/

Spent dialysate only

0

/

Blood compartment only

7

Pseudomonas spp.

Dialysate compartment only

9

Pseudomonas spp., P. mendocina,

Halomonas, Proteobacterium unc.

Whole blood/blood compartment

2

Pseudomonas spp.

Dialysate compartment/dialysate

2

Pseudomonas spp., P. mendocina

Different compartments (dialysate/blood)

14

Cannot be sequenced

Negative

4

/

Total

38

All the 3 sterile hemodialyzers (TORA 1 B3, NIPRO 190 E, TERUMO E 18) used as controls were negative both in the blood and dialysate compartments.

Bacterial DNA-positive and -negative patients were compared with inflammation data (table 4). Bacterial DNA-positive patients showed a trend toward an increase in hsCRP, IL-6, AOPP and a decrease in MFI DR+.

No significant results were obtained by comparing the groups divided as in table 4 according to the inflammatory data. But a trend was noticed in the following parameters for bacterial DNA patients when compared with bacterial DNA-negative patients: increases in hs-CRP, IL-6 and AOPP production and a reduction in MFI DR+. These data are indicative of an increase in inflammation (CRP), stimulation of mononuclear cells for proinflammatory cytokine production (IL-6), increase in oxidative stress by AOPP, and a decrease in immune response (MFI DR+) in the bacterial DNA-positive population.

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