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Saudi Journal of Kidney Diseases and Transplantation
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LETTER TO THE EDITOR  
Year : 2015  |  Volume : 26  |  Issue : 6  |  Page : 1300-1304
Circulating bacterial DNA fragments in chronic hemodialysis patients


1 Microbiology Department, High Institute of Public Health, Alexandria University, Alexandria, Egypt
2 Internal Medicine and Nephrology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

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Date of Web Publication30-Oct-2015
 

How to cite this article:
Hazzah WA, Hashish MH, El-Koraie AF, Ashour MS, Abbass AA. Circulating bacterial DNA fragments in chronic hemodialysis patients. Saudi J Kidney Dis Transpl 2015;26:1300-4

How to cite this URL:
Hazzah WA, Hashish MH, El-Koraie AF, Ashour MS, Abbass AA. Circulating bacterial DNA fragments in chronic hemodialysis patients. Saudi J Kidney Dis Transpl [serial online] 2015 [cited 2021 Oct 27];26:1300-4. Available from: https://www.sjkdt.org/text.asp?2015/26/6/1300/168689
To the Editor,

A number of bacterial products such as endotoxins, exotoxins and peptidoglycans, produced during bacterial growth and lysis, share the ability to induce cytokines and are known activators of immune functions. Bacterial DNA fragments (DNAb), oligodeoxynucleotides of six to 20 nucleotides, are newly recognized substances added to bacterial by-products that may pass dialyzer membranes. [1] Small-fragment DNAbs are ubiquitous contaminants even if dialysis fluids are subjected to strict sterilization procedures. [2],[3] A study evaluating whether bacterial DNA contamination might have an effect on apoptosis of activated monocytes from hemodialysis (HD) patients found that it enhances cytokine production and promotes the survival of inflammatory mononuclear cells. This raised a relevant issue of whether the presence of small amounts of DNAb will cause any significant biological response in the patient. [1],[4] It was shown that DNAb might contribute to the prolonged life span of inflammatory cells from HD patients, proposing that the presence of DNAb might cause both intensification and prolongation of the inflammatory response in HD patients. [4] The 16S ribosomal DNA (16S rDNA) gene is universal in bacteria and is present in multiple copies in the genomes of all known human bacterial pathogens that belong to the eubacterial kingdom. Using polymerase chain reaction (PCR) primers that are targeted at these conserved regions of rDNA, it is possible to design broad-range PCRs capable of detecting DNA from almost any bacterial species. [5] Several studies have considered that 16S rDNA gene PCR amplification is an optimal tool for the detection and identification of bacterial isolates. [3],[6],[7]

We conducted a cross-sectional study on 49 HD patients [24 and 25 from patients undergoing HD via central venous catheters (CVCs) and arteriovenous fistulae (AVF), respectively], chosen from two dialysis centers in Alexandria, Egypt. The present study aimed to assess whether bacterial DNA fragments are present in the blood of chronic HD patients potentially free of infection. After a thorough examination, patients having current infections or antibiotic intake within one week before sampling were excluded. Baseline clinical data, including age, gender, underlying renal disease, HD regimen, duration on dialysis and type of co-morbidities were recorded. Consents were taken before inclusion of patients in the study for detecting DNAb in blood samples and culturing withdrawn CVCs. In addition, ten healthy individuals were used as controls.

Whole blood samples were collected under sterile conditions from a peripheral vein before the start of the dialysis session. DNA was extracted from 200 μL of heparin-treated whole blood samples using a QIAamp ® DNA Blood Mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. Purified DNA was eluted with an elution buffer and was then ready for amplification.

Universal primers used for PCR amplification of the 16S rDNA gene were 355 F (5' CCTACGGGAGGCAGCAG-3') and 910 R (5'-CCCGTCAATTCCTTTGAGTT-3') (Operon Biotechnologies, Huntsville, USA ). [8] Aliquots of 10-μL DNA samples were used for amplification in a 50-μL PCR reaction mixture containing GoTaq® Green Master Mix (Promega, Madison, WI, USA). The temperature profile used for the amplification was 95°C for 5 min then 35 cycles of 95°C for 45 s, 53°C for 45 s and 72°C for 45 s and a final extension step of 7 min at 72°C. The amplification products (540 bp) were visualized by agarose gel electrophoresis and ethidium bromide staining.

Controls were included in each assay: Positive controls were prepared by inoculation of one blood sample by a laboratory isolated Escherichia coli (E. coli). DNA was extracted as previously described. Negative controls were prepared by the addition of nuclease-free water instead of the extract to the reaction mixture.

CVCs were withdrawn from 24 HD patients either for catheter malfunction or for replacement. Catheters (about 5 cm segment) were cultured by both the roll plate method [9] and vortexing [10] on both blood and MacConkey's agar plates. Following overnight aerobic incubation at 37°C, colonies were enumerated and calculated. Significant counts for catheter colonization were defined as more than 15 colony-forming units (CFUs)/catheter segment by roll plate or 100 CFUs/catheter segment by vortexing.

Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS version 15). Data are expressed as mean ± SD. P-values of <0.05 were considered significant. Characteristics of the 49 patients included in the study are given in [Table 1]. All healthy controls were negative for presence of bacterial DNA in the whole blood. PCR results varied with the frequency of HD sessions/ week and the type of vascular access, but did not show statistical significance. Considering the catheter colonization, 17 of 24 CVCs were colonized; however, there was no statistical difference in relation to the presence of DNAb in patients' blood. Of 49 HD patients, 30 (61.2%) were negative for DNAb, while 19 (38.8%) showed positive results [Table 2]. The mean duration of HD (1.6 ± 2.2 years vs 4.1 ± 3.3 years) was statistically significantly different between the groups.
Table 1: Characteristics of the 49 patients included in the study.

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Table 2: Relation between presence of bacterial DNA of 49 HD patients and different studied variables.

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A report by Schindler's et al was the first description of the occurrence of bacterial DNA fragments in clinically used solutions, especially dialysate. [1] They quantified the presence of these fragments in dialysate as a new type of dialysate contaminant and reported that DNA fragments were of sufficiently small size to pass through the dialyser membrane. Penetration of even a fraction of these DNAb through dialysis membranes onto the blood side is likely to have widespread effects on the immune system of dialysis patients. In view of that report, Serwanska-Swietek et al [11] investigated the presence of DNAb in 50 HD patients without any signs of active infection undergoing intermittent HD. DNAb was detectable in the blood of all studied patients, but they were not able to determine its source.

Reports of a different population of patients revealed that 80% of hemodialysis patients had trace amounts of DNA in serum. [12] Recently, Cazzavillan et al [9] investigated the presence of bacterial DNA in a population of HD patients in order to correlate the molecular data with the degree and level of inflammation and to evaluate its usefulness in the diagnosis of subclinical infection. DNAb was detected in either blood, dialysate or their compartments of the hemodialyzer. This finding was explained either by the bacteria or that DNAb might traverse the dialyser membrane, but there was no significant association between their presence and C-reactive protein and IL-6 levels.

Our study investigated the possible presence of DNAb in the blood of chronic HD patients free of any clinical signs of infection. Bacterial DNA was detected in 19 (38.8%) patients, while 30 (61.2%) patients showed negative results.

The significance of DNAb in the whole blood of dialysis patients is not yet determined. Considering the vascular access, the percentage of patients with circulating DNAb had a tendency to be less in patients with AVF than In patients with CVCs (regardless of being colonized or not), but showing no significant difference. This could be a result of bacterial colonization of the HD tubing system or due to contaminated dialysate. On other hand, the observation that 58.8% of cases with colonized CVCs were PCR negative might imply noninvasion or translocation of bacteria or DNAb from CVCs into the blood circulation. That brings us back to the term "significant colonization" of a catheter, because it does not always correlate with demonstrable clinical findings and not all catheters should be cultured unless infection is suspected.

The present study showed that higher the duration patients undergoing HD, chances of positive results of DNAb in their blood (P = 0.006) is increased. Moreover, although not proved to be statistically significant (P = 0.590), three HD sessions per week showed more positive results (42.1%) than two HD sessions/ week (27.3%). The question is how long the DNAb fragments circulate in a patient's blood or if it could be cumulative from each exposure to contaminated dialysate during the HD sessions and persist for the subsequent sessions as the samples in this study were taken just before the start of the dialysis sessions. Considering samples for checking dialysate contamination of the preceding session is not practical. Water and dialysate microbiological quality in both HD units were investigated on a routine basis. Although their results along the period of study complied with the acceptable limits (bacterial count <100 CFU/mL), the dialysate was not free of contamination. Thus, the source of DNAb is more likely to be endogenous or transferred from the dialysate of the preceding session. Repeated sampling to show whether DNAb was transient or persistent might have helped in this issue.

Little is known about plasma circulating bacterial DNA. Furthermore, the clearance mechanism of circulating DNA is poorly understood, although the liver and the kidneys play a major role for its removal. [7],[13] In the literature, the half-life of DNA in dead bacterial cells may vary greatly and is highly dependent on the environmental conditions. DNA persists in the tissues, as described for Borrelia burgdorferi DNA detected in the muscle of four of eight patients with chronic myalgia. [14] Also, Branger et al[15] detected DNA of Streptococcus pneumoniae from the cardiac valve of a man who had pneumococcal endocarditis seven years earlier, which aroused the question of persistence of DNAb without any evidence of infection.

Ratanarat et al [7] were investigating patients with severe sepsis undergoing continuous renal replacement therapy, and determined that DNAb was adsorbed within the hemofilter. In case of an undetectable amount of bacteria and DNAb from blood, this amount could be detected within the "concentrating" hemofilter. Interestingly, one of the studied patients, after three weeks of appropriate therapy, when the organism could not be detected by blood culture and DNAb in blood, DNAb still remained and was trapped in the blood compartment of the hemofilter. [7]

In conclusion, circulating DNAb fragments may be present in the blood of HD patients, but it is difficult to identify its source or persistence, entailing further investigations to trace its source and its long-term potential effect on patients. To the best of our knowledge, considering it as a tool for detection of bacteremia in HD patients receiving antibiotics or to confirm whether a colonized catheter is significantly of clinical interest is not clarified. Thus, the question of how to deal with patients having DNAb remains an issue to be further studied.

Conflict of interest: None declared.

 
   References Top

1.
Schindler R, Beck W, Deppisch R, et al. Short bacterial DNA fragments: Detection in dialysate and induction of cytokines. J Am Soc Nephrol 2004;15:3207-14.  Back to cited text no. 1
    
2.
Ledebo I. Ultrapure dialysis fluid - How pure is it and do we need it? Nephrol Dial Transplant 2007;22:20-3.  Back to cited text no. 2
    
3.
Gomila M, Gascó J, Gil J, Bernabeu R, Iñigo V, Lalucat J. A molecular microbial ecology approach to studying hemodialysis water and fluid. Kidney Int 2006;70:1567-76.  Back to cited text no. 3
    
4.
Navarro MD, Carracedo J, Ramírez R, et al. Bacterial DNA prolongs the survival of inflamed mononuclear cells in haemodialysis patients. Nephrol Dial Transplant 2007;22: 3580-5.  Back to cited text no. 4
    
5.
Harris KA, Hartley JC. Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol 2003;52 (Pt 8):685-91.  Back to cited text no. 5
    
6.
Warwick S, Wilks M, Hennessy E, et al. Use of quantitative 16S ribosomal DNA detection for diagnosis of central vascular catheter-associated bacterial infection. J Clin Microbiol 2004;42:1402-8.  Back to cited text no. 6
    
7.
Ratanarat R, Cazzavillan S, Ricci Z, et al. Usefulness of a molecular strategy for the detection of bacterial DNA in patients with severe sepsis undergoing continuous renal replacement therapy. Blood Purif 2007;25:10611.  Back to cited text no. 7
    
8.
Cazzavillan S, Ratanarat R, Segala C, et al. Inflammation and subclinical infection in chronic kidney disease: A molecular approach. Blood Purif 2007;25:69-76.  Back to cited text no. 8
    
9.
Maki DG, Weise CE, Sarafin HW. A semi-quantitative culture method for identifying intravenous-catheter-related infection. N Engl J Med 1977;296:1305-9.  Back to cited text no. 9
[PUBMED]    
10.
Brun-Buisson C, Abrouk F, Legrand P, Huet Y, Larabi S, Rapin M. Diagnosis of central venous catheter-related sepsis. Critical level of quantitative tip cultures. Arch Intern Med 1987;147:873-7.  Back to cited text no. 10
[PUBMED]    
11.
Serwanska-Swietek M, Popow A, Interewicz B, Olszewski R, Sikora M, Rydzewski A, eds. Bacterial DNA is Present in Blood of Hemodialysis Patients - Preliminary Report. 43rd ERA-EDTA Congress, Glasgow, UK; 2006.  Back to cited text no. 11
    
12.
Himmelfarb J, Stenvinkel P, Ikizler TA, Hakim RM. The elephant in uremia: Oxidant stress as a unifying concept of cardiovascular disease in uremia. Kidney Int 2002;62:1524-38.  Back to cited text no. 12
    
13.
Rhodes A, Wort SJ, Thomas H, Collinson P, Bennett ED. Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients. Crit Care 2006;10:R60.  Back to cited text no. 13
    
14.
Frey M, Jaulhac B, Piemont Y, et al. Detection of Borrelia burgdorferi DNA in muscle of patients with chronic myalgia related to Lyme disease. Am J Med 1998;104:591-4.  Back to cited text no. 14
    
15.
Branger S, Casalta JP, Habib G, Collard F, Raoult D. Streptococcus pneumoniae endocarditis: persistence of DNA on heart valve material 7 years after infectious episode. J Clin Microbiol 2003;41:4435-7.  Back to cited text no. 15
    

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Correspondence Address:
Dr. Walaa A Hazzah
Microbiology Department, High Institute of Public Health, Alexandria University, Alexandria
Egypt
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DOI: 10.4103/1319-2442.168689

PMID: 26586077

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