Stability and in vitro efficacy of antibiotic–heparin lock solutions potentially useful for treatment of central venous catheter-related sepsis

Jan C. Droste1, Hassan A. Jeraj2, Alan MacDonald3 and Ken Farrington1,*

1 Department of Renal Medicine, 2 Quality Control Laboratory and 3 Department of Microbiology, Lister Hospital, Stevenage, Herts SG1 4AB, UK

Received 7 December 2001; returned 26 May 2002; revised 2 January 2003; accepted 21 January 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Increasing numbers of patients for whom infection is a major risk are dependent on central venous catheters. Antibiotic–anticoagulant locks may have a role in preventing or treating catheter-related infections. The aim of this study was to determine the in vitro stability and efficacy of antibiotic–heparin lock solutions.

Methods: Candidate antibiotics (amikacin, ciprofloxacin, flucloxacillin, gentamicin, linezolid, teicoplanin) were investigated in vitro, either individually or in combination, in solution with heparin. The solutions were initially tested for visual precipitation. The efficacy of stable solutions and taurolidine was then tested in a catheter model bioassay system against microorganisms commonly encountered in catheter-related septicaemia.

Results: In general, lower concentrations of heparin (<=1000 U/mL) combined with antibiotics resulted in precipitation, whereas high concentrations (3500–10 000 U/mL) were compatible with a broader range of antibiotic concentrations. The stability of each antibiotic–heparin combination required individual assessment. Bioassays identified the following promising antibiotic–anticoagulant solutions: for broad-spectrum empirical cover, a teicoplanin–ciprofloxacin–heparin solution; for directed use, flucloxacillin–heparin for methicillin-susceptible Staphylococcus aureus (MSSA), high dose teicoplanin–heparin for methicillin-resistant S. aureus (MRSA), high-dose linezolid–heparin for vancomycin-resistant enterococci (VRE) and ciprofloxacin–heparin for (susceptible) Pseudomonas aeruginosa; for prophylactic use, taurolidine.

Conclusion: These solutions now warrant clinical trials to investigate their role in the management of catheter-related septicaemia.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the main challenges in the management of patients with indwelling central venous catheters (CVCs) for regular haemodialysis (HD) or long-term intravenous therapy is the high infection rate.18 CVCs are the most common source of hospital-acquired bacteraemia in hospitals in England,7 and current prophylaxis and treatment regimens are unsatisfactory. Catheter removal may not always be an option in chronic catheter dependency because of a lack of alternative vascular access sites. Treating CVC-related bacteraemia with systemic antibiotics without catheter removal often has only limited success,8,9 partly owing to biofilm formation within the CVC lumen. Biofilm formation is a relatively common occurrence,10 and the antibiotic resistance of organisms within a biofilm usually correlates with its maturity.11

The ‘antibiotic lock technique’,12,13 in which a concentrated antibiotic solution is instilled into the CVC lumen and allowed to dwell for several hours (TPN catheters) or days (HD catheters),5,14,15 looks promising. Most antimicrobial substances lack anticoagulant characteristics and hence need to be combined in antimicrobial–anticoagulant combinations. Taurolidine is an exception, combining both broad-spectrum antibacterial and anticoagulant properties.16,17 However, few studies have been performed in vitro to test the compatibility and stability of different antibiotics with anticoagulant.1820 Available data suggest that highly concentrated antibiotics tend to precipitate with low doses of heparin.21,22 Consequently, the optimal heparin concentration for antimicrobial–anticoagulant combinations is unknown.23

The aim of this study was to find physicochemically stable, antithrombotic, antibiotic lock solutions, which eliminate fully, in one treatment application, the intraluminal growth in a CVC model of Gram-positive and Gram-negative bacteria that commonly lead to catheter-related septicaemia. These are Staphylococcus epidermidis and Staphylococcus aureus in 59%, enteric Gram-negative bacilli including Pseudomonas in 17.9%, and enterococci and streptococci in 4.9% of episodes of catheter-related sepsis.4


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics and anticoagulants

All the antimicrobial solutions used in this study were prepared from their commercially available forms. These were: amikacin solution (paediatric: 50 mg/mL; Bristol-Myers Squibb, UK); ciprofloxacin solution (2 mg/mL; Bayer); flucloxacillin powder (CP Pharmaceuticals, Wrexham, UK); gentamicin solution (paediatric: 10 mg/mL; Hoechst Marion Roussel, Frankfurt, Germany); linezolid solution (2 mg/mL; Pharmacia Upjohn, UK); taurolidine (2%; Geistlich Pharma, Chester, UK); teicoplanin powder (Hoechst Marion Roussel); heparin sodium as monoparin (CP Pharmaceuticals); anticoagulant–citrate–dextrose solution (Sigma-Aldrich Chemie GmbH, Germany, C3821) with a composition of sodium citrate dihydrate (22.0 g/L), anhydrous citric acid (7.3 g/L) and glucose monohydrate (24.5 g/L).

Visual precipitation grid

Different concentrations of antibiotic–anticoagulant solutions were mixed in a grid of glass test tubes. The antibiotics and the heparin were diluted separately before combining them, in a total volume of 5 mL, to achieve the desired final antibiotic and heparin concentrations. The antibiotic–anticoagulant solutions were then incubated in the dark at 25 and 37°C (approximate catheter conditions, respectively, outside and inside the body). Visible precipitation was assessed immediately after preparation and after 24, 48 and 72–96 h and 7 days. All studies were performed in triplicate. Variability between triplicate samples was rare, but when it occurred the time quoted was that of the first tube to precipitate.

Bacterial strains

The bacterial strains employed were methicillin-susceptible S. aureus (MSSA) ATCC 6538, methicillin-resistant S. aureus (MRSA) No. MRQC 113, Pseudomonas aeruginosa ATCC 9027 and vancomycin-resistant Enterococcus faecalis (VRE) NCTC 12201 (PHLS). All these organisms are known to form biofilms.

Bioassay

CVC biofilms were established as described by Andris et al.24 Briefly, human blood, always from the same healthy volunteer, was instilled into the catheter to allow deposition of fibrin and other blood products on the CVC wall.25 The catheters used were polyurethane–silicone catheters (HFS 10/15, JOKA Kathetertechnik GmbH, Hechingen, Germany and KIMAL SL14F, Kimal, UK). After 10 min, the catheters were rinsed with normal saline and inoculated with bacteria in tryptic soy broth suspension (Oxoid, UK). The inoculated bacteria were harvested from overnight agar cultures into the broth, incubated for a further 3 h to obtain microorganisms in logarithmic growth cycle, and the final inoculum contained 108 cfu/mL. The catheters were incubated in the dark at 37°C for 24 h or 7 days to allow for bacterial colonization or biofilm production. No attempt was made to verify biofilm formation. After either 24 h or 7 days, antibiotic lock solutions were instilled into the catheters, which were then wrapped tightly with Parafilm M (American National Can, USA) at the catheter connector line–head junction (to prevent leakage on ultrasonication). The antibiotic lock solution was allowed to dwell for 48 h, following which the catheters were rinsed thoroughly with peptone water 1%, and ultrasonicated with the peptone water in the lumen. The intraluminal contents were flushed onto a tryptone soya agar plate. Following this, the parafilm was removed and the outer surface of the catheter wiped with alcohol and allowed to air-dry. Four 1-cm catheter segments were cut from each catheter. Two were immersed in broth and two were bisected, swabbed, and plated on tryptone soy agar (Oxoid, UK). Bacterial growth after 48 h was recorded for each of the three samples (from the flush after ultrasonication, from the catheter segments immersed in broth and from those bisected and swabbed). Growth occurring in any of these samples was recorded as a positive result.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Visual precipitation grid

Tables 13 show the results of the visual precipitation grid studies. In general, lower concentrations of heparin (<=1000 U/mL) resulted in antibiotic precipitation, whereas higher concentrations of heparin (3500–10 000 U/mL) were compatible with a wider range of antibiotic concentrations.


View this table:
[in this window]
[in a new window]
 
Table 1.  Testing grid summary for visual precipitation of ciprofloxacin–heparin solutions
 

View this table:
[in this window]
[in a new window]
 
Table 3.  Testing grid summary for visual precipitation of combined antibiotic and anticoagulant citrate dextrose
 
Most precipitates (over 90%) formed immediately, and once formed they did not re-dissolve. Exceptions were flucloxacillin and ciprofloxacin in heparin (Table 1, data only shown for ciprofloxacin) and teicoplanin–amikacin in heparin (Table 2). We found that flucloxacillin (20 mg/L) precipitated after 48 h at 25 and 37°C, when made up with water for injection or low doses of heparin, whereas at heparin concentrations of 4000 U/mL, solutions were stable for 72 h, although precipitation was present by 7 days. In previous studies, flucloxacillin in water for injection was found to be stable for 7 days at 20–25°C.26


View this table:
[in this window]
[in a new window]
 
Table 2.  Testing grid for visual precipitation of combined antibiotics and heparin
 
Teicoplanin (0.02–10 mg/mL) and linezolid (0.2– 1.92 mg/mL) were the only antibiotics that did not precipitate at any concentration when mixed with heparin. As a result, the precipitation grids are not shown for these agents.

The maximum stable concentrations of the different antibiotic components of a solution differed according to the anticoagulant solution used. Ciprofloxacin precipitated with heparin (Table 1) but not with citrate dextrose solution (data not shown) at the concentrations tested (0.2–0.8 mg/mL); however, the combination of teicoplanin and ciprofloxacin with citrate dextrose precipitated at higher antibiotic concentrations (Table 3). The maximum stable concentration of ciprofloxacin (0.4 mg/mL) in teicoplanin–ciprofloxacin anticoagulant citrate dextrose solution was only half that achievable in heparin, whereas the maximum stable concentration of teicoplanin (4 mg/mL) was the same in both anticoagulant solutions (Tables 2 and 3). It was possible to use a higherconcentration of teicoplanin with gentamicin in citrate dextrose (4 mg/mL) than in heparin solution (Tables 2 and 3). Temperature did not seem to affect the compatibility of antibiotic lock solutions.

Bioassay

The results of the bioassays are shown in Table 4. Growth was recovered from all the controls indicating bacterial colonization or biofilm production.


View this table:
[in this window]
[in a new window]
 
Table 4.  Antibiotic-lock solutions to eradicate growth in a CVC biofilm model
 
The combinations of teicoplanin (4 mg/mL), ciprofloxacin (0.8 mg/mL) and heparin (10 000 U/mL) (T4-CPX0.8-H10 000) showed the broadest spectrum of activity, including activity against P. aeruginosa and S. aureus (solution 1, Table 4). This combination was active against the 24 h biofilm of MRSA, and partly against the 7 day biofilm, where only two colony-forming units appeared on one agar plate after 48 h.

Ciprofloxacin 0.8 mg/mL with heparin (10 000 U/mL) (CPX0.8-H10 000), and with anticoagulant citrate dextrose (CPX0.8-ACD), eradicated P. aeruginosa from both 24 h and 7 day biofilms (solutions 2 and 15, Table 4). Lower doses of ciprofloxacin (CPX0.6-H4000), however, were either not sufficiently active against the 7 day biofilm, or failed to show any effect (CPX0.2-H4500) (solutions 9 and 10, Table 4).

Flucloxacillin–heparin (FX20-H4000; solution 3) was active against both the 24 h and 7 day biofilms of MSSA. Teicoplanin was only active against MRSA at a dose of at least 4 mg/mL; lower doses were ineffective even in combination with aminoglycosides (solutions 11–13). Inclusion of gentamicin in teicoplanin combinations appeared to enhance activity against MRSA, T4-H4500 (solution 7) having no effect on 7 day biofilm, unlike T4-G2-ACD (solution 14). There was also some enhancement of activity with ciprofloxacin and teicoplanin in this respect (T4-CPX0.8-H10 000 solution 1). Although higher concentrations of linezolid in heparin (LZ1.92-H2000; solution 4) were active against the 24 h biofilm of MRSA (7 day biofilm not tested), lower concentrations were not. Neither of the two lower linezolid combinations was effective against MRSA, either in heparin (LZ1.2-H2000) or citrate dextrose (LZ1.2-ACD) (solutions 8 and 18), although both were active against the 24 h biofilm of VRE. Only the higher concentration combination (LZ1.92-H2000) was active against the 7 day biofilm of VRE.

Combinations of teicoplanin, aminoglycosides and heparin (solutions 5 and 11–13) showed poor activity against the Pseudomonas strain, and only the combination of these agents with the high teicoplanin concentration (T10-A3-H3000; solution 5) was active against MRSA. Of the teicoplanin, aminoglycoside and citrate–dextrose combinations studied, T4-G2-ACD (solution 14) showed the broadest spectrum of activity, being active against 24 h biofilm of the Pseudomonas strain and against both the 24 h and 7 day biofilm of MRSA.

Taurolidine (solution 19) demonstrated the broadest activity of all antibiotic lock solutions, including activity against VRE, in line with previous MIC studies.16,27 However, taurolidine was only active against the 24 h biofilms and not the 7 day ones.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In general, precipitate formation was found to be concentration dependent, with higher concentrations of antibiotics requiring higher concentrations of heparin to prevent precipitation. Heparin is a heterogeneous mixture of variably sulphated polysaccharide chains, composed of repeating units of D-glucosamine and either L-iduronic acid or D-glucuronic acid.6 The pH of heparin solutions may vary between 5.5 and 8.0 for a 1% solution, depending on the batch of heparin used. Although the solubility of antibiotics in heparin may relate to a number of factors, including their pKa, the solubility product of any heparin–antibiotic salt that may be formed, and the possibility that heparin may form soluble complexes with antibiotics, it is probable that pH plays a major role.

Previous studies have suggested that some antibiotics, including gentamicin, are incompatible with heparin and cannot be used as antibiotic locks.12,28 This is not, however, supported by our results, although the concentrations at which antibiotic–heparin combinations precipitate appear unpredictable. Therefore we would suggest that each concentration of antibiotic, or antibiotic combination, and anticoagulant requires specific testing in vitro. Importantly, once a precipitate formed, it did not subsequently re-dissolve, which may be a significant safety issue, with immediate precipitation signalling incorrect preparation.

The prevalence of MRSA in hospitalized patients has increased dramatically in the past decade;7 that of VRE has also increased, but more modestly. It is therefore advisable to consider an infection with multiresistant bacteria early in any catheter-related septicaemia and to initiate empirical systemic antibiotic treatment with activity against a broad spectrum of microorganisms until the antibiogram is available. A number of studies have described the allocation of patients to either systemic treatment alone, systemic treatment plus antibiotic locks or antibiotic locks alone,3,8,9,12,14,15 with better outcome being achieved when both antibiotic lock and systemic treatment were used. The antibiotic lock combinations active against the broadest spectrum of bacteria in this study were the teicoplanin–ciprofloxacin–heparin solutions. However, for directed treatment in cases where the pathogen is either known or suspected, of the agents we tested, we would suggest flucloxacillin–heparin combinations for MSSA, teicoplanin–heparin or teicoplanin–gentamicin–heparin combinations for MRSA, linezolid–heparin combinations for VRE and ciprofloxacin–heparin combinations for P. aeruginosa, based on our results. Taurolidine displayed broad activity against 24 h, but not 7 day biofilms, and it may have a role in prophylaxis against catheter-related sepsis.13,17

Heparin is known to decrease CVC-related thrombosis,23 and although antibiotic locks without heparin have been used successfully in TPN catheters with a dwell time of <12 h,23 catheter occlusion was a risk.29 We would therefore suggest that when considering the antibiotic lock technique, heparin or an alternative anticoagulant should be used, especially when dwell times are prolonged. However, care is required since the use of high heparin concentrations (>5000 U/mL) may inadvertently result in systemic heparinization, especially in children, and patients of low body weight. At present, there is no consensus on the best dose of heparin to use in maintaining CVCs, with most dialysis centres using a dose ranging from 1000 to 10 000 U/ml,3,23,30 although TPN centres generally use lower concentrations.31,32 As there is the potential for antibiotics when used in lock solutions to reduce the activity of the antithrombotic agents, especially if precipitation occurs,22,33 we would suggest a heparin dose between 2000 and 10 000 U/mL as part of a stable antibiotic lock solution. Some centres use citrate dextrose in different strengths, or 3.13% trisodium citrate rather than heparin. Our studies do not suggest any major benefits of antibiotic-containing line-locks using citrate dextrose rather than heparin, although aminoglycoside solubility does appear to be improved. However, in general, combinations involving aminoglycosides performed poorly in our experiments to assess biofilm eradication and may not be the best agents for use in lock solutions.

In summary, we have shown that precipitate formation in antibiotic lock solutions is concentration dependent and that the stability of each antibiotic–anticoagulant combination needs to be assessed individually. We have identified a number of promising antibiotic lock solutions for empirical and directed treatment and prophylactic use in CVC-related infection. Although there are insufficient data on the long-term effects of antibiotic lock solutions on catheter integrity and durability or selection of resistant isolates, we feel that the antibiotic lock solutions identified in this study warrant clinical trials to investigate their role in both the treatment and prophylaxis of catheter-related infection.


    Acknowledgements
 
Many thanks to the staff in the Quality Control Laboratory and the Renal Unit of the Lister Hospital for their enthusiastic support for the practical aspects of this study.


    Footnotes
 
* Corresponding author. Tel: +44-1438-314-333 ext. 4230; Fax: +44-1438-781-174; E-mail: dr.farrington{at}lister.enherts-tr.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Powe, N. R., Jaar, B., Furth, S. L., Hermann, J. & Briggs, W. (1999). Septicemia in dialysis patients: incidence, risk factors, and prognosis. Kidney International 55, 1081–90.[CrossRef][ISI][Medline]

2 . Tokars, J. I., Miller, E. R., Alter, M. J. & Arduino, M. J. (1998). National surveillance of dialysis associated diseases in the United States, 1995. ASAIO Journal 44, 98–107.[ISI][Medline]

3 . Swartz, R. D., Boyer, C. L. & Messana, J. M. (1997). Central venous catheters for maintenance hemodialysis: a cautionary approach. Advances in Renal Replacement Therapy 4, 275–84.[Medline]

4 . Fletcher, S. J. & Bodenham, A. R. (1999). Catheter-related sepsis: an overview. British Society of Intensive Care Journal 2, 46–53.

5 . Domingo, P., Fontanet, A., Sanchez, F., Allende, L. & Vazquez, G. (1999). Morbidity associated with long-term use of totally implantable ports in patients with AIDS. Clinical Infectious Diseases 29, 346–51.[ISI][Medline]

6 . Vercaigne, L. M., Sitar, D. S., Penner, S. B., Bernstein, K., Wang, G. Q. & Burczynski, F. J. (2000). Antibiotic-heparin lock: in vitro antibiotic stability combined with heparin in a central venous catheter. Pharmacotherapy 20, 394–9.[ISI][Medline]

7 . Nosocomial Infection National Surveillance Service (2002). Surveillance of hospital-acquired infection in English hospitals 1997–2002. Public Health Laboratory, London, UK.

8 . Chawla, P. G. & Nevins, T. E. (2000). Management of hemodialysis catheter-related bacteremia–a 10-year experience. Pediatric Nephrology 14, 198–202.[CrossRef][ISI][Medline]

9 . 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]

10 . Tenney, J. H., Moody, M. R., Newman, K. A., Schimpff, S. C., Wade, J. C., Costerton, J. W. et al. (1986). Adherent microorganisms on lumenal surfaces of long-term intravenous catheters. Importance of Staphylococcus epidermidis in patients with cancer. Archives of Internal Medicine 146, 1949–54.[Abstract]

11 . Foley, I. & Gilbert, P. (1997). In-vitro studies of the activity of glycopeptide combinations against Enterococcus faecalis biofilms. Journal of Antimicrobial Chemotherapy 40, 667–72.[Abstract]

12 . Messing, B., Peitra-Cohen, S., Debure, A., Beliah, M. & Bernier, J. J. (1988). Antibiotic-lock technique: a new approach to optimal therapy for catheter-related sepsis in home-parenteral nutrition patients. Journal of Parenteral and Enteral Nutrition 12, 185–9.[ISI][Medline]

13 . Jurewitsch, B., Lee, T., Park, J. & Jeejeebhoy, K. (1998). Taurolidine 2% as an antimicrobial lock solution for prevention of recurrent catheter-related bloodstream infections. Journal of Parenteral and Enteral Nutrition 22, 242–4.[ISI][Medline]

14 . Krzywda, E. A., Andris, D. A., Edmiston, C. E., Jr & Quebbeman, E. J. (1995). Treatment of Hickman catheter sepsis using antibiotic lock technique. Infection Control and Hospital Epidemiology 16, 596–8.[ISI][Medline]

15 . Capdevila, J. A., Segarra, A., Planes, A. M., Ramirez-Arellano, M., Pahissa A., Piera, L. et al. (1993). Successful treatment of haemodialysis catheter-related sepsis without catheter removal. Nephrology Dialysis Transplantation 8, 231–4.[Abstract]

16 . Blenkharn, J. I. (1990). In-vitro antibacterial activity of noxythiolin and taurolidine. Journal of Pharmacy and Pharmacology 42, 589–90.[ISI][Medline]

17 . Reinmuller, J. (1999). The influence of taurolidine on physiological and pathological blood coagulation and implications for its use. Zentralblatt fur Chirurgie 124, Suppl. 4, 13–8.

18 . Henrickson, K. J., Powell, K. R. & Schwartz, C. L. (1988). A dilute solution of vancomycin and heparin retains antibacterial and anticoagulant activities. Journal of Infecious Diseases 157, 600–1.

19 . Regamey, C., Schaberg, D. & Kirby, W. M. (1972). Inhibitory effect of heparin on gentamicin concentrations in blood. Antimicrobial Agents and Chemotherapy 1, 329–32.[ISI][Medline]

20 . Jacobs, J., Kletter, D., Superstine, E., Hill, K. R., Lynn, B. & Webb, R. A. (1973). Intravenous infusions of heparin and penicillins. Journal of Clinical Pathology 26, 742–6.[ISI][Medline]

21 . Strong, D. K., Ho, W. & Nairn, J. G. (1989). Visual compatibility of vancomycin and heparin in peritoneal dialysis solutions. American Journal of Hospital Pharmacology 46, 1832–3.

22 . Tyler, L. S., Rehder, T. L. & Davis, R. B. (1981). Effect of gentamicin on heparin activity. American Journal of Hospital Pharmacology 38, 537–40.

23 . Randolph, A. G., Cook, D. J., Gonzales, C. A. & Andrew, M. (1988). Benefit of heparin in central venous and pulmonary artery catheters: a meta-analysis of randomized controlled trials. Chest 113, 165–71.

24 . Andris, D. A., Krzywda, E. A., Edmiston, C. E., Krepel, C. J. & Gohr, C. M. (1998). Elimination of intraluminal colonization by antibiotic lock in silicone vascular catheters. Nutrition 14, 427–32.[CrossRef][ISI][Medline]

25 . Merlino, R., Gaillard, J. L., Fauchere, J. L., Chaumont, P., Droy-Lefaix, M. T., Descamps, P. et al. (1988). In vitro quantitative model of catheter infection during simulated parenteral nutrition. Journal of Clinical Microbiology 26, 1659–64.[ISI][Medline]

26 . Lynn, B. (2002). Recent work on parenteral penicillins. Journal of Hospital Pharmacy 29, 183–94.

27 . Traub, W. H., Leonhard, B. & Bauer, D. (1993). Taurolidine: in vitro activity against multiple-antibiotic-resistant, nosocomially significant clinical isolates of Staphylococcus aureus, Enterococcus faecium, and diverse Enterobacteriaceae. Chemotherapy 39, 322–30.[ISI][Medline]

28 . Easom, A. (2000). Prophylactic antibiotic lock therapy for hemodialysis catheters. Journal of Nephrology Nursing 27, 75.

29 . . Johnson, D. C., Johnson, F. L. & Goldman, S. (1994). Preliminary results treating persistent central venous catheter infections with the antibiotic lock technique in pediatric patients. Pediatric Infectious Disease Journal 13, 930–1.[ISI][Medline]

30 . Trivedi, H. S. & Twardowski, Z. J. (1997). Use of double-lumen dialysis catheters. Loading with locked heparin. ASAIO Journal 43, 900–3.[ISI][Medline]

31 . Cottee, S. (1995). Heparin lock practice in total parenteral nutrition. Professional Nurse 11, 25–9.

32 . Bailey, M. J. (1979). Reduction of catheter-associated sepsis in parenteral nutrition using low-dose intravenous heparin. British Medical Journal 1, 1671–3.[Medline]

33 . Koup, J. R. & Gerbracht, L. (1975). Reduction in heparin activity by gentamicin. Drug Intelligence and Clinical Pharmacy 9, 568.