In situ measurement of linezolid and vancomycin concentrations in intravascular catheter-associated biofilm

Mark H. Wilcox,*, Peter Kite, Kerry Mills and Sarah Sugden

Department of Microbiology, University of Leeds and The General Infirmary, Leeds LS2 9JT, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We describe a new method for the measurement of antimicrobial concentrations in the biofilm associated with the endoluminal surface of intravascular catheters. We quantified endoluminal planktonic bacteria in haemodialysis catheters using the acridine orange method on catheter blood. After catheter removal, separate lumens were perfused in vitro with either vancomycin or linezolid to simulate in vivo antibiotic infusion. Biofilm was recovered using endoluminal brushes, weighed and assayed by fluoroimmunoassay for vancomycin and by bioassay for linezolid. Viable bacteria were counted by serial dilution and agar plating. Biofilm had measurable amounts of vancomycin in 11/11 catheter lumens post-infusion (0.3–18.2 mg biofilm per lumen, mean 6.8 mg; vancomycin concentration 0.2–89 mg/g biofilm, median 19 mg/g). By comparison, linezolid was detected in 4/11 catheter lumens post-infusion (0.5–18.1 mg biofilm per lumen, mean 5.9 mg; linezolid concentration 0.9–6.1 mg/g biofilm, median 1.5 mg/g). Percentage reductions in biofilm-associated bacterial counts post-antibiotic were 84–100%, median 95% (vancomycin) and 0–98%, median 91% (linezolid). We found a significant difference (P = 0.05; Wilcoxon rank sum test) in vancomycin concentrations in coagulase-negative staphylococcal biofilm (median 17.0 mg/g, mean 27.9 mg/g) compared with glycopeptide levels found in biofilm associated with other microorganisms (median 5.5 mg/g, mean 6.9 mg/g). Biofilm concentrations of vancomycin are generally higher than linezolid after antibiotic infusion, which can be explained partly by glycopeptide binding to glycocalyx. Neither antibiotic achieved consistent 100% kill of biofilm bacteria after single infusions, even when a very high concentration was present. The endoluminal brush technique can be used to measure antibiotic concentration in intravascular catheter-associated biofilm in situ. This approach can be exploited to measure biofilm antibiotic concentrations in vivo, without the need for catheter removal.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Use of intravascular catheters is compromised by infection, which, for example, affects 3–7% of central venous catheters (CVCs).1 While catheter removal is often advocated as a means of removing the suspected or proven focus of infection, it is common instead to leave catheters in situ and use antimicrobial salvage therapy, in order to preserve bloodstream access. It is pertinent to note that the great majority of CVCs removed on clinical suspicion of catheter-related bloodstream infection (CR-BSI) subsequently prove not to yield significant numbers of bacteria on laboratory testing.2,3 As catheter-associated infection is characterized by biofilm accumulation, primarily on the endoluminal surfaces of catheters,4 antimicrobial agents must first penetrate glycocalyx in order to reach and kill embedded microbes.5 Also, a slow metabolic state renders biofilm microorganisms less susceptible to antimicrobials than planktonic microbes.6,7 However, few data are available to quantify antibiotic concentrations in biofilm,7 and none that we are aware of concern those associated with intravascular catheters, principally because of the inaccessibility of luminal material.

We have described the clinical use of the endoluminal brush to sample CVC biofilm,8,9 to provide an accurate laboratory diagnosis of CR-BSI that does not rely on catheter removal. The present study aimed to exploit this approach to recover endoluminal biofilm for antimicrobial assay. We first validated the technique in vitro and then sought to compare the concentrations of linezolid and vancomycin in CVC biofilm following antibiotic infusion. We also compared the activity of these two antibiotics against CVC-associated, slow-growing, biofilm bacteria by comparing microbial counts pre- and post-infusion.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We confirmed the presence of Gram-positive bacterial endoluminal biofilm in vivo in five polyurethane CVCs (Nipro, Horizon Medical Products, GA, USA) used for haemodialysis, dwell time 1–14 days (median 8 days), using the Gram/acridine orange leucocyte cytospin (G/AOLC) technique, with blood aspirated through the catheters.10,11 Briefly, a 1 mL sample of blood, collected in a tube containing EDTA, was aspirated in vivo from the catheter lumen for the G/AOLC test. Each of two 50 µL samples was mixed with formalin–saline, followed by centrifugation at 352g for 5 min. The homogenized cellular deposit was transferred into a cytospin cupule containing a microscope slide. The cellular suspension was centrifuged at 153g for 5 min in a cytocentrifuge (Shandon, Runcorn, UK). A monolayer of leucocytes and microorganisms was created on each of two microscope slides, which were stained with either 1:10 000 w/v acridine orange or Gram's stain, and viewed by ultra-violet (UV) or light microscopy. A minimum of 100 high-powered fields were examined and the presence of any microorganisms within the cellular monolayer (on either slide) was considered a positive result. Also, a quantitative colony count was carried out on 10 and 100 µL aliquots of the catheter blood sample. Bacterial isolates were identified by standard techniques and using API identification strips. Colonized CVCs used throughout the study were removed when no longer clinically required.

Following their removal, catheters were perfused separately through each lumen (n = 8) in vitro with vancomycin (1 g in 250 mL of 5% dextrose) for 2 h to simulate glycopeptide administration in vivo. Each catheter lumen was then flushed with 10 mL of 0.9% saline to remove non-biofilm-associated antibiotic. Endoluminal biofilm was recovered from each catheter lumen by passing an endoluminal brush from the hub to the tip.8 Biofilm was recovered from brushes by vortexing for 30 s in 1 mL of 0.9% saline. Brushes were weighed before and after each procedure, and the wet weight of recovered biofilm was determined (blotting paper was used to remove excess saline). Vancomycin concentration in solubilized biofilm (in the presence and absence of streptokinase) was then determined by fluoroimmunoassay (Abbott, UK). The lower limit of sensitivity for this assay was 1 mg/L. Manipulations of solubilized biofilm were minimized to reduce potential binding of vancomycin to plastic surfaces.12

We collected 11 further polyurethane haemodialysis catheters (dwell time 3–64 days, median 9 days) associated with Gram-positive bacterial endoluminal biofilm as described above. After catheter removal, separate lumens were perfused in vitro with either vancomycin (1 g in 250 mL of 5% dextrose) or linezolid (600 mg in 300 mL of 5% dextrose) for 2 h. Linezolid concentrations were measured by bioassay, using Staphylococcus aureus NCTC 6571 as the indicator strain. The lower limit of sensitivity for this assay was 2 mg/L. Biofilm vancomycin concentrations were measured as above. Remaining viable biofilm-associated bacteria were enumerated by serial dilution and agar plating of 10 and 100 µL aliquots of each solubilized biofilm.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the first series of CVCs studied, biofilm containing Gram-positive bacteria (quantitative sampling of blood taken for the G/AOLC test yielded Staphylococcus epidermidis, Staphylococcus aureus or enterococci) had measurable amounts of vancomycin in 8/8 samples of solubilized biofilm post-antibiotic infusion (0.4–4.9 mg biofilm per lumen, mean 2.5 mg) (TableGo). The concentration of vancomycin recovered ranged from 1.0 to 17.3 mg/g biofilm (mean 9.8 mg/g). Inclusion of streptokinase during biofilm solubilization did not increase the recovery of vancomycin (data not shown).


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Table. Results of CVC endoluminal sampling and biofilm vancomycin assay
 
In the second series of CVCs, biofilm had measurable amounts of vancomycin in 11/11 catheter lumens postinfusion (0.3–18.2 mg biofilm per lumen, mean 6.8 mg; vancomycin concentration 0.2–89 mg/g biofilm, median 19 mg/g). By comparison, linezolid >2 mg/L was detected in 4/11 samples of solubilized biofilm post-infusion (0.5– 18.1 mg biofilm per lumen, mean 5.9 mg; linezolid concentration 0.9–6.1 mg/g). Percentage reductions in biofilm-associated bacterial counts post-vancomycin were 84–100%, median 95%. Corresponding figures post-linezolid were 0–98%, median 91% (FigureGo).



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Figure. (a) The effect of vancomycin infusion on biofilm-associated viable count. (b) The effect of linezolid infusion on biofilm-associated viable count. Full data sets available for eight antibiotic–biofilm exposures. Zero values are converted to ‘1’ to allow logarithmic plot.

 
After combining data from both series, there was a significant difference (P = 0.05, Wilcoxon rank sum test) in the vancomycin concentration in coagulase-negative staphylococcal biofilm (median 17.0 mg/g, mean 27.9 mg/g) compared with glycopeptide levels found in biofilm associated with other microorganisms (median 5.5 mg/g, mean 6.9 mg/g). This variance was not significantly related to differences in either CVC median dwell time (14 versus 7.5 days) or biofilm median weight (3.1 versus 3.5 mg).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well accepted that antibiotic activity against sessile bacteria is reduced compared with planktonic cells, and that this relates to decreased specific growth rate and growth phase.6,13,14 However, resuspension of biofilms leads to increased antibiotic susceptibility,6 suggesting that glycocalyx may impair biofilm penetration or inhibit antibiotic activity. The effect of bacterial biofilm on antibiotic activity was elegantly demonstrated using two isogenic strains of S. epidermidis.15 The parent strain grew on surfaces in multiple layers and this promoted phenotypic resistance to antibiotic treatment with levofloxacin, rifampicin or teicoplanin. Killing of a mutant strain, which adhered but did not accumulate, was significantly greater in vitro and in an animal model. Darouiche et al.7 measured vancomycin concentrations in S. epidermidis biofilm growing in vitro on stainless steel. They found that bacterial growth in biofilm was inversely related to vancomycin concentration in biofilm, but even extremely high levels did not eradicate biofilm bacteria. Vancomycin MICs and MBCs for biofilm bacteria did not differ from those for control microorganisms. They concluded that the failure of glycopeptides to cure prosthesis-related infection is probably due to diminished antimicrobial effect on biofilm bacteria as opposed to poor antibiotic penetration. Vancomycin levels in biofilms reported by Darouiche et al.7 (up to 1.2 mg/g) were less than, but of similar magnitude to, those recorded in the present study. These authors used vancomycin concentrations up to 0.16 mg/mL compared with 4 mg/mL in our study. Also, biofilms were produced by broth-cultured S. epidermidis, which characteristically yields excess glycocalyx compared with the relatively scanty material formed by bacteria grown in body fluids.16,17 Evans and Holmes18 found no significant differences between vancomycin-exposed and control groups in S. epidermidis biofilm viable and total cell counts after 10 days. MICs and MBCs of vancomycin for the original S. epidermidis strain were 3.125 and 6.25 mg/L, respectively. However, for biofilm suspensions, although vancomycin MICs were 3.125 mg/L, MBCs were >400 mg/L.

This study is the first to measure antibiotic concentrations in CVC-associated biofilm formed in vivo. We found that vancomycin concentrations in biofilm varied markedly. For example, the concentration of vancomycin in S. aureus biofilm ranged from 1 to 22 mg/g. Nevertheless, the median biofilm vancomycin concentration grossly exceeded the MIC for Gram-positive cocci. However, Ceri et al.,19 using an in vitro model designed to determine the minimal biofilm eradication antibiotic concentrations, found that exposure to vancomycin 1024 mg/L still did not eradicate biofilm staphylococci. Notably, on all except one occasion we were unable to achieve complete killing of biofilm-associated bacteria, despite exposure to actual biofilm vancomycin concentrations thousands of times higher than the expected MICs of Gram-positive cocci. These data provide strong evidence that the failure of antibiotics to kill biofilm bacteria is not due to lack of drug penetration.

Linezolid concentrations in biofilm were frequently below the lower limit of assay sensitivity, and were usually less than respective vancomycin levels in biofilm from adjacent lumens. This is unlikely to be due to the difference in antibiotic concentration infused (vancomycin 4 mg/mL, linezolid 2 mg/mL). There are several alternative explanations for this observation. Linezolid may penetrate biofilm poorly, but this is unlikely given its generally high levels in tissue20,21 and its activity at reducing the viable count in most biofilms examined here. Flushing after antibiotic infusion, to remove non-biofilm-associated antibiotic, may have removed linezolid that had diffused into biofilm. This latter possibility could be related to a difference between linezolid and vancomycin in their ability to bind to biofilm material.

We found significantly higher vancomycin levels (three-fold difference in median concentrations) in coagulase-negative staphylococcal biofilm compared with those in biofilm associated with other bacteria. This is the first evidence in situ that vancomycin binds to coagulase-negative staphylococcal biofilm. Vancomycin binds to a polysaccharide-containing extract of S. epidermidis slime,21 which is hyper-produced by most highly adherent strains. Indeed, such binding was found to reduce the activity of vancomycin and teicoplanin, but not clindamycin, rifampicin or cefazolin, in a concentration-dependent manner against all 18 isolates examined.22 The authors concluded that impaired glycopeptide activity by slime may explain why these antibiotics are sometimes ineffective in eradicating coagulase-negative staphylococcal biofilm infections. Our in situ observations are consistent with these earlier in vitro data. It appears therefore that vancomycin, but not linezolid, binds to biofilm glycocalyx associated with coagulasenegative staphylococci. It is not possible to be certain whether the lack of affinity of linezolid with biofilm glycocalyx is a desirable attribute, or indeed how linezolid will compare with glycopeptides for the treatment of biofilm-associated infections. In theory, for an antibiotic to be particularly effective against biofilm-associated microorganisms it should have affinity for glycocalyx, but not be inhibited by such binding, and be cidal against slowly dividing cells. Such attributes may be unobtainable in vivo for one antibiotic.

We were unable to demonstrate a correlation between CVC dwell time and antibiotic concentration in biofilm. For the reasons stated above, we would expect that there should be a correlation between biofim vancomycin concentration and number of coagulase-negative staphylococci before exposure, but due to the number of catheter lumens examined we were unable to confirm this assumption. Similarly, it was not possible to determine whether vancomycin or linezolid penetration alters with the age of the biofilm. Nevertheless, killing of biofilm bacteria was generally greater after vancomycin exposure than after linezolid. Whether this difference would remain after multiple, as opposed to single, infusions remains unknown. Although linezolid is bacteriostatic, in phase II/III studies it has shown similar efficacy to bactericidal antibiotics in vivo,23,24 possibly due to better pharmacokinetics such as excellent tissue penetration.20,21 Vancomycin is weakly bactericidal both in vitro and in vivo.2527

We have established that the endoluminal brush technique can be used to measure antibiotic concentration in intravascular catheter-associated biofilm in situ. These data can be extended to investigate specific antibiotic–microorganism interactions within biofilm, and to determine the effects of multiple antibiotic infusions. Furthermore, given the good safety data during use of the endoluminal brush,28 the technique may be exploited to measure drug penetration and accumulation in biofilm in vivo, without the need for catheter removal. Success rates employing antimicrobial therapy but not catheter removal for CR-BSI are disappointing.29,30 Using the endoluminal brush, it should be possible to determine whether in vivo killing of biofilm microorganisms has occurred. In turn, comparison of antimicrobial agents could permit the selection of more efficacious drugs for the treatment of CR-BSI, possibly increasing the success of catheter non-removal approaches.


    Acknowledgments
 
We thank the staff on the Renal Unit at Leeds General Infirmary for their help with the collection of catheters, and Pharmacia Corporation for providing financial support for the study.


    Notes
 
* Corresponding author. Tel: +44-113-233-5596; Fax: +44-113-233-5649; E-mail: markwi{at}pathology.leeds.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Maki, D. G. (1994). Infections caused by intravascular devices used for infusion therapy: pathogenesis, prevention, and management. In Infections Associated with Indwelling Medical Devices, 2nd edn, (Bisno, A. I & Waldvogel, F. A., Eds), pp. 155–212. ASM Press, Washington, DC.

2 . Ryan, J. A., Abel, R. M., Abbott, W. M., Hopkins, C. C., Chesney, T. M., Colley, R. et al. (1974). Catheter complications in total parenteral nutrition; a prospective study of 200 consecutive patients. New England Journal of Medicine 290, 757–61.[ISI][Medline]

3 . Padberg, F. T. Jr, Ruggiero, J., Blackburn, G. L. & Bistrian, B. R. (1981). Central venous catheterization for parenteral nutrition. Annals of Surgery 193, 264–70.[ISI][Medline]

4 . Dobbins, B., Kite, P. & Wilcox, M. H. M. (1999). Diagnosis of central venous catheter related sepsis—a critical look inside. Journal of Clinical Pathology 52, 165–72.[Free Full Text]

5 . Dibdin, G. H., Assinder, S. J., Nichols, W. W. & Lambert, P. A. (1996). Mathematical model of ß-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released beta-lactamases. Journal of Antimicrobial Chemotherapy 38, 757–69.[Abstract]

6 . Duguid, I. G., Evans, E., Brown, M. R. & Gilbert, P. (1992). Effect of biofilm culture upon the susceptibility of Staphylococcus epidermidis to tobramycin. Journal of Antimicrobial Chemotherapy 30, 803–10.[Abstract]

7 . Darouiche, R. O., Dhir, A., Miller, A. J., Landon, G. C., Raad, I. I. & Musher, D. M. (1994). Vancomycin penetration into biofilm covering infected prostheses and effect on bacteria. Journal of Infectious Diseases 170, 720–3.[ISI][Medline]

8 . Kite, P., Dobbins, B. M., Wilcox, M. H., Fawley, W. N., Kindon, A. J., Thomas, D. et al. (1997). Evaluation of a novel endoluminal brush for the in-situ diagnosis of catheter related sepsis. Journal of Clinical Pathology 50, 278–82.[Abstract]

9 . Tighe, M. J., Kite, P., Fawley, W. N., Thomas, D. & McMahon, M. J. (1996). An endoluminal brush to detect the infected central venous catheter in situ: a pilot study. British Medical Journal 313, 1528–9.[Free Full Text]

10 . Rushforth, J. A., Hoy, C. M., Kite, P. & Puntis, J. W. (1993). Rapid diagnosis of central venous catheter sepsis. Lancet 342, 402–3.[ISI][Medline]

11 . Kite, P., Dobbins, B. M., Wilcox, M. H. & McMahon, M. J. (1999). Rapid diagnosis of central venous catheter related blood stream infection without catheter removal. Lancet 354, 1504–7.[ISI][Medline]

12 . Wilcox, M. H., Winstanley, T. G. & Spencer, R. C. (1994). Binding of teicoplanin and vancomycin to polymer surfaces. Journal of Antimicrobial Chemotherapy 33, 431–41.[Abstract]

13 . Evans, D. J., Brown, M. R., Allison, D. G. & Gilbert, P. (1990). Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle. Journal of Antimicrobial Chemotherapy 25, 585–91.[Abstract]

14 . Desai, M., Bühler, T., Weller, P. H. & Brown, M. R. (1998). Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth. Journal of Antimicrobial Chemotherapy 42, 153–60.[Abstract]

15 . Schwank, S., Rajacic, Z., Zimmerli, W. & Blaser, J. (1998). Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics. Antimicrobial Agents and Chemotherapy 42, 895–8.[Abstract/Free Full Text]

16 . Wilcox, M. H., Hussain, M., Faulkner, M. K., White, P. J. & Spencer, R. C. (1991). Slime production and adherence by coagulase-negative staphylococci. Journal of Hospital Infection 18, 327–32.[ISI][Medline]

17 . Hussain, M., Wilcox, M. H. & White, P. J. (1993). The slime of coagulase-negative staphylococci: biochemistry and relation to adherence. FEMS Microbiology Reviews 107, 191–207.

18 . Evans, R. C. & Holmes, C. J. (1987). Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrobial Agents and Chemotherapy 31, 889–94.[ISI][Medline]

19 . Ceri, H., Olson, M. E., Stremick, C., Read, R. R., Morck, D. & Buret, A. (1999). The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. Journal of Clinical Microbiology 37, 1771–6.[Abstract/Free Full Text]

20 . Stalker, D. J., Wajszczuk, C. P. & Batts, D. H. (1997). Linezolid safety, tolerance and pharmacokinetics after intravenous dosing twice daily for 7.5 days. In Program and Abstracts of the ThirtySeventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract A116. p. 23. American Society for Microbiology, Washington, DC.

21 . Chiba, K., Feenstar, K. L., Slatter, J. G., Daley-Yates, P., Duncan, J. N., Fagerness, P. E. et al. (1998). Absorption, distribution, metabolism, and excretion of the oxazolidinone antibiotic linezolid (PNU-100766) in the Sprague Dawley rat. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract A123. p. 39. American Society for Microbiology, Washington, DC.

22 . Farber, B. F., Kaplan, M. H. & Clogston, A. G. (1990). Staphylococcus epidermidis extracted slime inhibits the antimicrobial action of glycopeptide antibiotics. Journal of Infectious Diseases 161, 37–40.[ISI][Medline]

23 . Duvall, S., Bruss, J., Todd, W. & Hafkin, B. (2000). Linezolid in the treatment of staphylococcal skin and soft tissue infections: Combined results from three phase III multinational clinical trials. Clinical Microbiology and Infection 6, Suppl. 1, 64, Abstract WeP20.

24 . Cammarata, S. K., San Pedro, G. S., Timm, J. A., Hempsall, K. A., Todd, W. M., Oliphant, T. H. et al. (2000). Comparison of linezolid versus ceftriaxone/cefpodoxime in the treatment of hospitalized patients with community-acquired pneumonia. Clinical Microbiology and Infection 6, Suppl. 1, 136. Abstract WeP237.

25 . Marr, K. A., Kong, L., Fowler, V. G., Gopal, A., Sexton, D. J., Conlon, P. J. et al. (1998). Incidence and outcome of Staphylococcus aureus bacteremia in hemodialysis patients. Kidney International 54, 1684–9.[ISI][Medline]

26 . Tam, V. H., Jumbe, N. L., Briceland, L. L. & Miller, M. H. (1999). A comparative study of vancomycin and ß-lactams in the treatment of staphylococcal bacteremia in haemodialysis patients. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 1095. p. 723. American Society for Microbiology, Washington, DC.

27 . Perry, J. D., Jones, A. L. & Gould, F. K. (1999). Glycopeptide tolerance in bacteria causing endocarditis. Journal of Antimicrobial Chemotherapy 44, 121–4.[Abstract/Free Full Text]

28 . Dobbins, B. M., Kite, P., Wilcox, M. H., Kindon, A. J. L., Tighe, M. J. & McMahon, M. J. (1997). Clinical safety of the endoluminal brush technique for in-situ diagnosis of catheter related sepsis. In Program and Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract J189. p. 323. American Society for Microbiology, Washington, DC.

29 . Marr, K. A., Sexton, D. J., Conlon, P. J., Corey, G. R., Schwab, S. J. & Kirkland, K. B. (1997). Catheter-related bacteremia and outcome of attempted catheter salvage in patients undergoing hemodialysis. Annals of Internal Medicine 127, 275–80.[Abstract/Free Full Text]

30 . O'Riordan, E. & Conlon, P. J. (1998). Haemodialysis catheter bacteraemia: evolving strategies. Current Opinion in Nephrology and Hypertension 7, 639–42.[ISI][Medline]

Received 21 July 2000; returned 25 September 2000; revised 4 October 2000; accepted 17 October 2000