J Antimicrob Chemother 1997; 39: 285â286. Alan P. Johnson. Antibiotic Reference ...... Hindler4 and Gould et al.5 described quinolone-resistant. H. influenzae ...
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Journal of Antimicrobial Chemotherapy (1997) 39, 285–296
Correspondence Veterinary use of antimicrobial agents and problems of resistance in human bacterial infections J Antimicrob Chemother 1997; 39: 285–286 Alan P. Johnson Antibiotic Reference Unit, Laboratory of Hospital Infection, Central Public Health Laboratory, London NW9 5HT, UK
Sir, The recent Leading Article by Dr Piddock on the veterinary use of antimicrobial agents1 was timely, and mirrors current concern about this practice. Further data not included in this article, but which are pertinent to the debate about the spread of antimicrobial resistance from animals to man, have been derived from studies of the emergence of resistance to apramycin, an aminoglycoside antibiotic which is licensed solely for veterinary use. Apramycin was introduced into veterinary medicine in the early 1980s, and is used primarily in calves and pigs for the treatment of diarrhoea and other infections caused by Gram-negative bacteria. Investigators in several countries including the UK, France and Belgium have shown that apramycin-resistant bacteria can be isolated from animals, particularly those treated with apramycin.2–5 Several studies have shown that the mechanism of resistance involves production of the aminoglycosidemodifying enzyme 3-N-aminoglycoside acetyltransferase type IV (AAC[3]IV).3,4 This enzyme has a broad substrate specificity, with the result that bacteria producing it are not only resistant to apramycin, but show crossresistance to gentamicin and other aminoglycosides used to treatserious infections in humans. The finding that the veterinary use of apramycin could promote the carriage and faecal excretion of aminoglycoside-resistant bacteria by farm animals, gave rise to concern that these resistant bacteria might spread to humans. The justification for this concern was confirmed when Threlfall et al.6 reported apramycin resistance associated with cross-resistance to gentamicin in Salmonella typhimurium phage type 204c isolated from humans.Subsequently, apramycin/gentamicin-resistant human clinical isolates belonging to other species, including Escherichia coli and Klebsiella pneumoniae, were reported from various countries including the UK,7 Eire,8 Belgium5 and
Spain.9 The mechanism of resistance usually involved production of AAC(3)IV and molecular studies indicated that the genes encoding the enzyme were of veterinary origin.7,9 Further evidence supporting the passage of resistant organisms from animals to humans was provided by the isolation of apramycin-resistant E. coli from the faeces of a pig handler and his wife who worked on a farm where an outbreak of diarrhoea caused by apramycin-resistant E. coli occurred in a pig unit.4 The above studies should reinforce the message that control of the spread of antibiotic resistance requires the prudent use of antibiotics not only in the field of human medicine but also in veterinary medicine.
References 1. Piddock, L. J. V. (1996). Does the use of antimicrobial agents in veterinary medicine and animal husbandry select antibiotic-resistant bacteria that infect man and compromise antimicrobial chemotherapy? Journal of Antimicrobial Chemotherapy 38, 1–3. 2. Chaslus-Dancla, E., Glupczynski, Y., Gerbaud, G., Lagorce, M., Lafont, J. P. & Courvalin, P. (1989). Detection of apramycin resistant Enterobacteriaceae in hospital isolates. FEMS Microbiology Letters 61, 261–6. 3. Chaslus-Dancla, E., Pohl, P., Meurisse, M., Marin, M. & Lafont, J. P. (1991). High genetic homology between plasmids of human and animal origins conferring resistance to the aminoglycosides gentamicin and apramycin. Antimicrobial Agents and Chemotherapy 35, 590–3. 4. Hunter, J. E. B., Bennett, M., Hart, C. A., Shelley, J. C. & Walton, J. R. (1994). Apramycin-resistant Escherichia coli isolated from pigs and a stockman. Epidemiology and Infection 112, 473–80. 5. Pohl, P., Glupczynski, Y., Marin, M., Van Robaeys, G., Lintermans, P. & Couturier, M. (1993). Replicon typing characterization of plasmids encoding resistance to gentamicin and apramycin in Escherichia coli and Salmonella typhimurium isolated from human and animal sources in Belgium. Epidemiology and Infection 111, 229–38. 6. Threlfall, E. J., Rowe, B., Ferguson, J. L. & Ward, L. R. (1986). Characterization of plasmids conferring resistance to gentamicin and apramycin in strains of Salmonella typhimurium phage type 204c isolated in Britain. Journal of Hygiene 97, 419–26. 7. Johnson, A. P., Burns, L., Woodford, N., Threlfall, E. J., Naidoo, J., Cooke, E. M. et al. (1994). Gentamicin resistance in clinical isolates of Escherichia coli encoded by genes of veterinary origin. Journal of Medical Microbiology 40, 221–6. 8. Johnson, A. P., Malde, M., Woodford, N., Cunney, R. J. & Smyth, E. G. (1995). Urinary isolates of apramycin-resistant Escherichia coli and Klebsiella pneumoniae from Dublin. Epidemiology and Infection 114, 105–12.
285 © 1997 The British Society for Antimicrobial Chemotherapy
Correspondence 9. Salauze, D., Otal, I., Gomez-Lus, R. & Davies, J. (1990). Aminoglycoside acetyltransferase 3-IV (aacC4) and hygromycin B 4-1 phosphotransferase (hphB) in bacteria isolated from human and animal sources. Antimicrobial Agents and Chemotherapy 34, 1915–20.
‘Cost-effectiveness’—is it always effective? J Antimicrob Chemother 1997; 39: 286 I. Dorrian, G. S. Tillotson and R. M. Lee Pharmaceutical Division, Bayer plc, Strawberry Hill, Newbury, Berkshire RG14 1JA, UK Sir, With current financial constraints in the National Health Service, physicians and pharmacists are combining their skills, intellect and effort to find ways of providing effective therapy by cost-effective means. Since the realization that some antibiotics, more notably the fluoroquinolones, have excellent and predictable bioavailability when administered orally, the use of the oral route has increased. Indeed, drugs such as ciprofloxacin and ofloxacin have been shown to be as effective for the treatment of severe systemic infections when administered orally instead of parenterally.1–3 Financial drivers have caused us to appreciate the additional costs incurred when drugs are given parenterally,4 thus further increasing the cost element of treating infections. Although, in many instances, this change is laudable for both financiers and patients it is wrong to believe that one can replace parenteral administration with oral medication in a wholesale fashion. It has come to our attention that an increasing number of intensive care and other units are endeavouring to save money by giving ciprofloxacin tablets crushed in a solution via a nasogastric tube. The patients are also likely to be receiving a plethora of other pharmaceutical products such as enteral feeds, prophylaxis for stress ulcers, etc. It is our responsibility as a pharmaceutical company to ensure that our products are being used optimally, thus we would like to highlight one of the potentially deleterious interactions which could lead to ciprofloxacin being misused. There is evidence to show that when ciprofloxacin is given concomitantly with enteral feeds the maximum serum concentration is decreased; this occurs whether the two products are given orally or as tablets crushed and injected via a G-tube.5 Similar findings have also been observed with ofloxacin.6 Undoubtedly this is due to the recognized interaction between divalent cations such as calcium, iron and zinc which are present in these enteral feeds.7 The degree of this decreased absorption is of the order of halving the serum and tissue levels. Clearly when using ciprofloxacin in the intensive care setting for serious infections one needs adequate concentrations of the drug to inhibit the
normally problematic pathogens. However, if one is confident that the patient is not receiving any contra-indicated concomitant medications which are listed in the product data sheet, then it is reasonable to be cost-effective by administering ciprofloxacin orally at a dose of either 500 mg or 750 mg depending on the likely pathogens. We hope that there will be fewer failures of therapy among patients who should be receiving these drugs parenterally. In the overall scheme of things iv antibiotics for a few days could save a prolonged stay on an intensive care unit due to giving oral antibiotics cheaply but less effectively.
References 1. Fass, R. J. (1987). Efficacy and safety of oral ciprofloxacin for treatment of serious urinary tract infections. Antimicrobial Agents and Chemotherapy 31, 148–50. 2. Powers, T. & Bingham, D. H. (1990). Clinical and economic effect of ciprofloxacin as an alternative to injectable antimicrobial therapy. American Journal of Hospital Pharmacy 47, 1781–4. 3. Cooke, J., Cairns, C. J., Tillotson, G. S., Conner, S., Lewin, S. K. M., Nicholls, J. et al. (1993). Comparative clinical, microbiologic, and economic audit of the use of oral ciprofloxacin and parenteral antimicrobials. Annals of Pharmacotherapy 27, 785–9. 4. Kerr, J. R., Barr, J. G. & Smyth, E. T. M. (1993). Computerised calculation of the true costs of antibiotic therapy. European Journal of Clinical Microbiology and Infectious Diseases 12, 622–5. 5. Healy, D. P., Brodbeck, M. & Clendening, C. (1994). Ciprofloxacin absorption impaired by enteral feedings given orally and via G-tube in patients. In Abstracts of the Thirty-Fourth Inter science Conference on Antimicrobial Agents and Chemotherapy, Orlando, FL, 1994. Abstract A35, p. 18. American Society for Microbiology, Washington, DC. 6. Mueller, B. A., Brierton, D. G., Abel, S. R. & Bowman, L. (1994). Effect of enteral feeding with ensure on oral bioavailabilities of ofloxacin and ciprofloxacin. Antimicrobial Agents and Chemother apy 38, 2101–5. 7. Nix, D. E. (1993). Drug–drug interactions with fluoroquinolone antimicrobial agents. In Quinolone Antimicrobial Agents, 2nd edn (Hooper, D. C. & Wolfson, J. S., Eds), pp. 245–58. American Society for Microbiology. Washington, DC.
Why do patients with infection remain in hospital once changed to oral antibiotics? J Antimicrob Chemother 1997; 39: 286–288 Anne C. Boyterb, John Stephena, Peter G. Fegana and Dilip Nathwania a
Infection and Immunodeficiency Unit and bDepartment of Pharmacy, Dundee Teaching Hospitals, NHS Trust, King’s Cross Hospital, Dundee DD3 8EA, UK Sir, Health care systems are currently being reorganized as they simultaneously try to improve quality of patient care
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Correspondence and reduce costs. The prescribing of antimicrobials is an important target for those attempting to achieve these goals, particularly as antibiotics account for between 3% and 25% of all prescriptions,1 and up to 50% of the drug budget in hospitals.2 A significant proportion of these drug costs is due to use of expensive broad-spectrum parenteral agents, often inappropriately prescribed.3 Traditionally, patients with serious sepsis are admitted to hospital for intravenous antibiotics as physicians have been sceptical of treating serious infections with oral antibiotics, primarily due to concern over efficacy. However, the past decade has witnessed an expansion of oral antimicrobials which have excellent pharmacokinetic and pharmacodynamic properties, equivalent efficacy to iv antimicrobials and good safety profiles.4,5 This has allowed clinicians and pharmacists to employ many strategies aimed at reducing the use of parenteral agents. One such strategy has been an early switch from iv to oral, or sequential therapy, allowing early patient discharge. The proponents of this strategy highlight the clinical advantages, economic benefits and the benefit to the patient and health care purchaser.6,7 However, despite the fact that the total cost of a day in hospital significantly outweighs the direct drug acquisition costs, the policy of observing hospitalized patients for a minimum of 1 day after switching them from iv therapy for common uncomplicated infections is widely prevalent and is not based on scientific evidence. Indeed, a recent study highlighted the limited usefulness of an in-hospital observation period for patients who had received iv antibiotics for pyelonephritis.8 The economic benefits accrued by avoiding an extra day of hospitalization in the USA were estimated to amount to millions of dollars annually.9 This practice of in-patient observation appears to be common for other infections treated with parenteral agents.8 We retrospectively reviewed the case notes of patients who had received iv antibiotics and were subsequently changed to oral therapy, in an attempt to understand and improve our own clinical practice, particularly with a view to patient discharge. This retrospective study was carried out on patients admitted to the Infection and Immunodeficiency Unit at King’s Cross Hospital, Dundee in the 6-month period from January to August 1994. All patients who had received an iv antimicrobial were suitable for inclusion in the study and were identified from ward records. A random selection of these case records were reviewed. Data collected included: demographics, total length of hospital stay, duration of iv and oral therapy, main clinical indication for prescribing the antibiotic, documented reason(s) for the patient remaining in hospital once parenteral antimicrobials had been discontinued and the oral therapy instigated. A record of which day the patient was converted to oral therapy was also made. Evidence of clinical relapse during the time on oral therapy was defined as recurrence of fever, worsening of clinical
symptoms or signs, any abnormal result such as a new positive culture or documentation of an antibiotic-related adverse drug reaction. Of the 129 patients admitted to the Infection and Immunodeficiency Unit in the 6-month period from January to August 1994, who received parenteral anti-infective therapy, 86 sets of case records were randomly sampled and reviewed. Forty patients were male. The median age was 46 years (interquartile range 31–72 years). The majority (74.4%) were direct referrals from their General Practitioners and 82.6% had had no previous anti-infective therapy. The indications for anti-infective therapy are detailed in Table I. The most common indications for parenteral therapy were soft tissue infections, pneumonia and urinary tract infections. Patients remained on parenteral therapy for a median of 3 days (interquartile range 2–4 days) and had a median total length of treatment of 10 days (interquartile range 8–12 days). The median length of stay in hospital was 7 days (interquartile range 4–11 days). Therefore, patients were in hospital, usually on oral antibiotics, for a median of 4 days before hospital discharge. Twenty-two of 86 patients (25.5%) had a specific reason(s) for staying in hospital after completion of parenteral therapy. These are summarized in Table II. We assume that 64/86 (74.4%) of patients were kept in Table I. Main indications for parenteral antimicrobial therapy in 86 hospitalized patients Indication
Number
Soft tissue infection Lower respiratory tract infection Meningitis Urinary tract infection Bacterial tonsillitis Severe or disseminated herpes zoster Severe sepsis of unknown aetiology Cholangitis Other
22 15 3 16 9 5 10 3 4
Table II. Indications for continued stay in hospital Indication
Number
Persisting temperature Worsening clinical progress Adverse drug reaction Requiring further investigation Non-infection-related medical problems Social
12 2 1 3 8 6
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Correspondence hospital for observation or for other reasons which could not be ascertained from the case records. It was of interest to note that the majority (70/86) of decisions to change to oral therapy coincided with the Consultant ward round, indicating that senior members of staff were usually involved in making the decision to change to oral therapy. Only 15/86 (17%) had infection- or antibiotic-related documented reasons for continuing to be in hospital. These 15 patients made an uncomplicated recovery despite persisting fever or worsening of clinical progress. Although this study did not include contacting patients after they were discharged from hospital, it is significant that only 2/86 (2.3%) were re-admitted with an infection within one month of discharge. The single adverse drug reaction was minor and would not normally require hospitalization. The remaining 7/86 (8%) had noninfection-related medical or social reasons necessitating hospitalization. Ten patients (11.6%) had both an infection-related and a non-infection-related reason for their continued stay. This study, which examines the reasons for continuing hospitalization in patients who have received parenteral antimicrobial therapy for a variety of infections, reveals that 74.4% of patients were kept in hospital for what we assume to be purely for observation after changing to oral therapy. However, it is possible that there were other reasons not clearly stated in the notes. Of the patients kept in hospital for either continuing fever or relapse, none required return to iv therapy and all made a full recovery. Non-infection-related medical and social problems accounted for the main reasons for continued stay in hospital. We conclude that in an adult population with a heterogeneous group of infections the policy of continuing hospitalization as a means of observing patients once they have been converted to oral therapy should be reviewed. These patients should be offered early hospital discharge with attention paid to production of clear pre-discharge planned arrangements for further review by the primary care clinician or hospital out-patient clinic. As a consequence of this survey these measures are already being implemented within our own unit. The impact of this policy will be subject to further audit. We urge others in similar situations to review their own clinical practice critically, as patient and economic benefits are worth considering when the average cost per case of treating a patient with an infectious disease in a unit similar to ours, in Scotland (1994/95) was nearly £2000, a significant proportion of which is due to in-patient stay.
References 1. Col, N. F. & O’Connor, R. W. (1987). Estimating worldwide current antibiotic usage: report of Task Force 1. Reviews of Infectious Diseases 9, Suppl. 3, S232–43.
2. Barriere, S. L. (1985). Cost-containment of antimicrobial therapy. Drug Intelligence and Clinical Pharmacy 19, 278–81. 3. Kunin, C. M., Tupasi, T. & Craig, W. A. (1973). Use of antibiotics: a brief exposition of the problem and some tentative solutions. Annals of Internal Medicine 79, 555–60. 4. Craig, W. A. & Andes, D. R. (1995). Parenteral versus oral antibiotic therapy. Medical Clinics of North America 79, 497–508. 5. Bartlett, J. G. (1995). Impact of new oral antibiotics on the treatment of infectious diseases. Infectious Diseases in Clinical Practice 4, Suppl. 2, S50–7. 6. Guay, D. R. P. (1993). Sequential antimicrobial therapy. A realistic approach to cost-containment. PharmacoEconomics 3, 341–4. 7. Mandell, L. A., Bergeron, M. G., Gribble, M. J., Jewesson, P. J., Low, D. E., Marrie, T. J. et al. (1995). Sequential antibiotic therapy: effective cost management and patient care. Canadian Journal of Infectious Diseases 6, 306–15. 8. Caceres, V. M., Stange, K. C., Kikano, G. E. & Zyzanski, J. (1994). The clinical utility of a day of hospital observation after switching from intravenous to oral antibiotic therapy in the treatment of pyelonephritis. Journal of Family Practice 39, 337–9. 9. National Center for Health Statistics. (1991). Detailed Diagnoses and Procedures, National Hospital Discharge Survey, 1989. Vital and Health Statistics, Series 13, No 108. US Department of Health and Human Services, Hyattsville, MD.
The use of Amphotericin B Lipid Complex J Antimicrob Chemother 1997; 39: 288–290 Ian M. Franklina, Jayesh Mehtab and Tim Rootb a
Bone Marrow Transplant Unit, Glasgow Royal Infirmary, Glasgow G4 0SF; bThe Royal Marsden Hospital, London SW3 6JJ, UK. Sir, Amphotericin B is a naturally occurring polyene macrolide antifungal agent produced by strains of Streptomyces nodosus and was first identified in 1953. Even after so many years in clinical use, it remains the treatment of choice for opportunistic fungal infections. Intravenous use, however, is limited by its nephrotoxicity. In recent years, attempts have been made to reduce this by the development of sophisticated formulations which include amphotericin B in liposomes and lipid complexes and allow use of higher doses and/or continuation of treatment for longer. Three such novel formulations are currently licensed in the UK. Whilst these products certainly appear to offer advantages over conventional amphotericin B, this has been achieved at the expense of much higher direct cost. In today’s cash-limited environment, it becomes increasingly important to define those situations in which clinical need justifies the use of such products. In order to
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Figure. Treatment algorithm for use of ABLC
overcome any local bias in developing such guidelines, it is useful to develop as wide a consensus as possible, both geographically and professionally. We have, therefore, developed the attached algorithm (Figure) to help clinicians decide when it is appropriate to use one of these new lipid-based products, Amphotericin B Lipid Complex ABLC; Abelcet. In doing so, it is not our intention to recommend or endorse the use of any one product over another, but to give an indication of how this product may best be used. There are particular problems in developing guidelines for systemic antifungal therapy as patients are by definition already seriously ill and treatment is usually there-
fore initiated before a fungus has been positively identified. The most common starting point is an immunosuppressed or neutropenic patient with a pyrexia of unknown origin who has not responded to appropriate antibiotic treatment for at least 72 h. Data from experimental infections have shown that ABLC is either as effective or slightly less effective than conventional amphotericin B on a milligram for milligram basis.1,2 As ABLC is given at a dose of 5 mg/kg/day, as compared with 1 mg/kg/day with conventional amphotericin, it seems likely that the higher tissue concentrations it achieves will be effective in situations where conventional amphotericin has failed.3 This view is
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Correspondence supported by the results of recent trials comparing ABLC with conventional amphotericin in the treatment of aspergillosis4 and candida infections.5 Usually, neutropenic patients start overcoming their fungal infections when their neutrophil counts recover, but this is not always the case. The degree of neutropenia is therefore an important factor in determining therapy. Confirmation of invasive fungal infection is also important and a definitive diagnosis should be vigorously pursued. Lipid-based amphotericin B treatment should be considered when there is highly suspected or confirmed invasive fungal infection in a neutropenic or immunosuppressed patient. Renal failure is the most common dose-related toxicity of conventional iv amphotericin B and is a frequent cause of premature discontinuation of treatment. If renal function starts to deteriorate, the discontinuation of all other nephrotoxic drugs or their substitution by appropriate but less toxic alternatives should obviously be considered but may not always be possible. If renal function falls below an acceptable level (e.g. a serum creatinine concentration above 170 mol/L and rising, or twice the pre-treatment baseline level) consideration should be given to replacing conventional amphotericin B by a lipid-based formulation. This course of action is supported by a recent study of patients with invasive mycosis in which 61 of 228 patients started with a baseline serum creatinine concentration above 225 mol/L. In this group, renal function actually improved whilst patients were receiving ABLC for up to 6 weeks.6 Potassium requirement may be so great in patients receiving conventional amphotericin B that the serum potassium level can no longer be maintained at an acceptable level (e.g. 2.5 mmol/L) and this is also an indication for considering a change to a lipid-based formulation. The less serious, yet unpleasant, side effects of conventional amphotericin B such as rigors and chills, if uncontrolled by appropriate prophylaxis, may also be a reason for changing to such a formulation. Once treatment has been started with ABLC, it is important to continue to monitor the patient and their renal function, to watch for signs of infusion-related events and to take appropriate action should these occur. Unfortunately, there are still insufficient data from controlled trials to determine precisely the clinical situations in which the new amphotericin formulations are likely to be of most value and in which their cost can therefore readily be justified. There is also a danger in the overrigid enforcement of any prescribing policy as clinical decisions have to be made on an individual basis. Nevertheless, having recognized these provisos, we hope that the above guidelines and the algorithm (Figure) will be useful to those considering the use of ABLC or other lipid-based formulations.
References 1. Clark, J. M., Whitney, R. R., Olsen, S. J., George, R. J., Swerdel, M. R., Kunselman, L. et al . (1991). Amphotericin B lipid complex therapy of experimental fungal infections in mice. Antimicrobial Agents and Chemotherapy 35, 615–21. 2. Perfect, J. R. & Wright, K. A. (1994). Amphotericin B lipid complex in the treatment of experimental cryptococcal meningitis and disseminated candidosis. Journal of Antimicrobial Chemotherapy 33, 73–81. 3. Janknegt, R., de Marie, S., Bakker-Woudenberg, I. A. J. M. & Crommelin, D. J. A. (1992). Liposomal and lipid formulations of amphotericin B. Clinical Pharmacokinetics 23, 279–91. 4. Hiemenz, J. W., Lister, J., Anaissie, E. J., White, M. H., Dinubile, M., Silber, J. et al. (1995). Emergency-use amphotericin B lipid complex (ABLC) in the treatment of patients with aspergillosis: historical control comparison with amphotericin B. Blood 86, Suppl. 1, 849a. 5. Anaissie, E. J., White, M., Uzun, O., Singer, C., Bodey, G. P., Matzke, D. et al. (1995). Amphotericin B lipid complex versus amphotericin B for treatment of hematogenous and invasive candidiasis: a prospective, randomized, multicenter trial. In Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Abstract LM21, p. 330. American Society for Microbiology, Washington, DC. 6. Walsh, T. J., Hiemenz, J. W., Seibel, N. & Anaissie, E. J. (1994). Amphotericin B lipid complex in the treatment of 228 cases of invasive mycosis. In Abstracts of the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, FL, 1994. Abstract M69, p. 247. American Society for Microbiology, Washington, DC.
In-vitro pharmacodynamic simulation of clavulanic acid concentrations: effect on Staphylococcus aureus and Haemophilus influenzae -lactamase activity J Antimicrob Chemother 1997; 39: 290–292 M. Martína, L. Aguilarb*, I. P. Balcabaoa, M. L. Gómez-Lusa, R. Dal-Réb and J. Prietoa a
Microbiology Department, School of Medicine, Universidad Complutense, Madrid; bMedical Department, SmithKline Beecham Pharmaceuticals, Valle de la Fuenfria no. 3, 28034 Madrid, Spain *Correspondence: Tel: 34-1-3345275; Fax: 34-1345141. Sir, The post- -lactamase inhibitor effect is the effect on the viability of organisms of the removal of -lactamase inhibitor.1 We studied the in-vitro effect of simulated serum concentrations of clavulanic acid (after a 125 mg dose)2 on the viability and -lactamase activity of Staphy -
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Correspondence lococcus aureus NCTC 11561 and Haemophilus influenzae ATCC 35056. Clavulanic acid MICs were 125 and 32 mg/L for S. aureus and H. influenzae respectively. Overnight cultures were diluted in Mueller–Hinton broth (Difco, Detroit, MI, USA) for S. aureus or Haemophilus Test Medium broth3 for H. influenzae and incubated until an absorbance of 0.3 units (580 nm, spectrophotometer Hitachi U-100) was achieved. Cultures were further diluted 1:10 thus obtaining final inocula of 8.5 106 cfu/mL and 107 cfu/mL for S. aureus and H. influenzae respectively in 4 mL broth containing the first clavulanic acid concentration (Table). Incubation was performed in Centriprep-10 (10 kDa membrane pore size) concentrator tubes (Amicon Ltd, Gloucestershire, UK) at 37°C in a shaking water bath, for the first incubation period shown in the Table. An aliquot of 200 L was diluted 10-fold and, in triplicate, 20 L were plated on agar. Centrifugation–filtration at 2000g for 10 min was performed and the supernatant above the filter was collected for -lactamase activity determination. One hundred microlitres of S. aureus and 150 L of H. influenzae suspensions remained below the filter. New broths (4 mL) with the next clavulanic acid concentration were added to the bacterial suspensions and vortex agitation was performed. Tubes were incubated for the next incubation period. This methodology was performed throughout seven incubation periods, four times for each strain, simultaneously controlled with tubes without clavulanic acid. Clavulanic acid carry-over effect, yielded by the remaining bacterial suspensions after the centrifugation– filtration, was calculated using Y 0.025X for S. aureus and Y 0.375X for H. influenzae, where X is the initial clavulanate amount in 4 mL and Y the resulting clavulanate in 100 or 150 L. Final concentrations (Table) were calculated by adding Y to the new broth concentrations for each incubation period. -Lactamase activity was measured by adding 25 L of a 500 mg/L nitrocefin solution (Glaxo Ltd, Greenford, Middlesex, UK) to 225 L of each broth supernatant, which were then incubated for 30 min at 37°C. Afterwards, 1.5 mL of phosphate buffer 0.05M were added and absorbance (482 nm) was read in the spectrophotometer, using broth without inoculum as the baseline absorbance. Intergroup comparisons of viability (Student’s t-test) and -lactamase activity (ANOVA) were performed. Due to multiple comparisons a P 0.01 was defined as statistically significant. No differences (P 0.01) were found between centrifugation–filtration control experiments and standard growth curves. Differences (P 0.001) in viability were found between clavulanic acid and controls from 2 h to 12 h for S. aureus and from 2 h to 10 h for H. influenzae. Differences (P 0.001) in -lactamase activity were found between clavulanic acid and controls from 3 h onwards for both strains. The significant decrease in 291
Correspondence -lactamase activity relative to the control was maintained despite the increase in viability, even after 8 h when clavulanic acid was not present. If we define the post -lactamase effect as a significant difference in -lactamase activity relative to the control after clavulanic acid removal, a value of at least 4 h (from 8 to 12 h) for both strains was obtained. In agreement with other authors4 we found that clavulanic acid sub-inhibitory concentrations delayed the S. aureus generation time. S. aureus penicillinase reactivation began 2 h after disappearance of clavulanic acid, never reaching control -lactamase activity. H. influenzae TEM -lactamase activity remained significantly below baseline values from 3 h onwards. The decrease in staphylococcal -lactamase activity relative to the control for at least 12 h and the decrease in TEM -lactamase activity relative to the baseline for the same interval could affect dosing strategies for -lactamase inhibitor/ -lactam combinations, provided similar results are obtained with a panel of bacteria exhibiting varying degrees of susceptibility to these combinations.
Acknowledgements This work was supported by a grant from Smith Kline Beecham Pharmaceuticals, Madrid, Spain. We thank M. J. Giménez and F. Soriano for their critical review.
References 1. Thorburn, C. E. & Molesworth, S. J. (1992). The post -lactamase inhibitor effect (PLIE): a novel aspect of the activity of clavulanic acid in antibacterial tests. In Program and Abstracts of the Thirty-Second Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, CA, 1992. Abstract 540, p. 201. American Society for Microbiology, Washington, DC. 2. Adam, D., de Visser, I. & Koeppe, P. (1982). Pharmacokinetics of amoxicillin and clavulanic acid administered alone and in combination. Antimicrobial Agents and Chemotherapy 22, 353–7. 3. Jorgensen, J. H., Redding, J. S., Maher, L. A. & Howell, A. W. (1987). Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. Journal of Clinical Microbiology 25, 2105–13. 4. Gillissen, G. & Hasse, G. (1987). L’acide clavulanique. Son effet sur le staphylocoque doré outre l’effet inhibiteur sur les betalactamases. Pathologie Biologie 35, 503–6.
Isolation and characterization of a ciprofloxacin-resistant isolate of Haemophilus influenzae from The Netherlands J Antimicrob Chemother 1997; 39: 292–293 H. J. Bootsmaa,b , A. Troelstrac, A. van VeenRutgersa,F. R. Mooia, A. J. de Neelinga* and B. P. Overbeekc a
Research Laboratory for Infectious Diseases, RIVM, PO Box 1, 3720 BA, Bilthoven; bEijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Utrecht; cSt Antonius Hospital, Nieuwegein, The Netherlands *Corresponding author
Sir, Bacterial infections are a recurrent problem in patients with chronic obstructive pulmonary disease. The bacterial pathogens most likely to be involved in communityacquired respiratory infections in these patients are Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Among these species resistance to empirically administered antimicrobial therapy occurs, and this can be the cause of treatment failure. An 83-year-old male patient with a history of severe emphysema who had been subjected to bilobectomy in 1981 for carcinoma of the lung, was admitted to hospital because of an exacerbation of his obstructive pulmonary disease in October 1995. He had been treated for several days with cefaclor without clinical improvement. Microbiological examination of the purulent sputum showed, per microscopic field, 20–50 leucocytes, 0–10 Grampositive cocci and 50–100 Gram-negative bacilli. The bacilli were remarkably long and slim and therefore morphologically suspect for a Pseudomonas species. Antimicrobial therapy was started with ciprofloxacin orally 500 mg twice daily. The next day Klebsiella oxytoca and H. influenzae were grown in culture. The isolate of H. influenzae (strain no. 24482) was confirmed as biovar IV, serotype e by Dr F. A. G. Reubsaet (RIVM, Bilthoven). Susceptibility tests were done according to NCCLS guidelines1 by the agar-dilution method with haemophilus test agar (Oxoid), an inoculum of 104 cfu/spot and 20 h incubation at 35 C and in 5% CO2. H. influenzae Rd, kindly donated by Dr L. van Alphen (Academic Medical Centre, Amsterdam), was used as a reference (Table). Strain 24482 was resistant to ciprofloxacin and to cotrimoxazole, but susceptible to the -lactam antibiotics and to tetracycline. Absence of clinical improvement combined with these laboratory findings resulted in a switch to co-amoxiclav 625 mg three times daily po. The
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Correspondence Table. MIC (mg/L) of ciprofloxacin and other antibiotics for H. influenzae 24482 and H. influenzae Rd
Antibiotic
MIC (mg/L) strain 24482 Rd strain
Ciprofloxacin Co-trimoxazole Amoxycillin Co-amoxiclav Cefuroxime Tetracycline
2 16/304 0.25 0.25/0.12 2 0.25
These identical substitutions in E. coli suggest that the resistance of our H. influenzae is due to the amino-acid substitutions at positions 84 and 88 of its gyrA-protein.
References
0.008 0.12/2.28 1 0.5/0.25 0.5 0.25
1. National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Third Edition; Approved Standard M7-A3. NCCLS, Wayne, PA.
clinical condition of the patient improved and he was discharged several days later. Resistance to the newer quinolones in H. influenzae is rare. In a large Canadian study in 1992 and 1993, all of 1688 strains were susceptible to ciprofloxacin.2 Jones found 1% of 7961 H. influenzae isolates to be moderately susceptible,3 and 1% resistant to lomefloxacin. Barriere & Hindler4 and Gould et al.5 described quinolone-resistant H. influenzae from patients with chronic respiratory tract infections. In an ongoing electronic surveillance in Dutch public health laboratories, only 0.19% of 2678 single isolates were resistant to ciprofloxacin (MIC 1 mg/L). Therefore, strain 24482is one of the first ciprofloxacinresistant H. influenzaeisolates in the Netherlands. Since mutations in the DNA gyrase (gyrA) gene are the most common mechanism of fluoroquinolone resistance in other bacterial species,6 we sequenced the corresponding region of the gyrA gene of the H. influenzae strains Rd and 24482. DNA was extracted by heating a suspension of cells (106 cfu/mL) for 15 min at 100 C and 10 L of each sample was subjected to a ‘touchdown’ PCR7 with an annealing temperature ranging from 61 C to 50 C. Based on the gyrA sequence present in GenBank (accession number U32806) primers were developed corresponding to residues 43 to 25 (For; 5 -ATGCTATAATCCGCCACAA-3 ) and 536–515 (Rev; 5 -ATCCCCACCGCAATACCAGAAG-3 ) of the gyrA gene. DNA sequencing of both strands of the PCR products was performed in duplicate with an ABI 373A DNA sequencer according to the instructions provided by the manufacturer (Applied Biosystems, Foster City, CA, USA) using the same primers. The sequence of the Rd strain (positions 1–510) was identical to the sequence obtained from the database. The sequence of the quinolone-resistant isolate (positions 1–510, deposited in GenBank, accession number Z73213) differed in 11 bases. Eight of these replacements were silent and only three mutations resulted in amino acid substitutions, in codon 84 (TCC TTA; Ser Leu) and codon 88 (GAT TAT; Asp Tyr). Homologous double mutations have been found in codons 83 and 87 of several isolates of ciprofloxacin-resistant Escherichia coli. 6
2. Scriver, S. R., Walmsley, S. L., Kau, C. L., Hoban, D. J., Brunton, J., McGeer, A. et al . (1994). Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their -lactamases. Canadian Haemophilus Study Group. Antimicrobial Agents and Chemotherapy 38, 1678–80. 3. Jones, R. N. (1992). Fluoroquinolone (Lomefloxacin) International Surveillance Trial: a report of 30 months of monitoring in vitro activity. American Journal of Medicine 92, Suppl. 4A, 52–7S. 4. Barriere, S. L. & Hindler, J. A. (1993). Ciprofloxacin-resistant Haemophilus influenzae infection in a patient with chronic lung disease. Annals of Pharmacotherapy 27, 309–10. 5. Gould, I. M., Forbes, K. J. & Gordon, G. S. (1994). Quinolone resistant Haemophilus influenzae. Journal of Antimicrobial Chemotherapy 33, 187–8. 6. Vila, J., Ruiz, J., Marco, F., Barcelo, A., Goñi, P., Giralt, E. et al. (1994). Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrobial Agents and Chemotherapy 38, 2477–9. 7. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K. & Mattick, J. S. (1991). “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Research 19, 4008.
Azithromycin uptake by tissue cultured epithelial cells J Antimicrob Chemother 1997; 39: 293–295 Alvaro Pascual*, Jesus Rodriguez-Baño, Sofía Ballesta, Isabel García and Evelio J. Perea Department of Microbiology, School of Medicine, University of Seville, Apdo 914, Seville 41080, Spain *Tel:
34-5-4557448; Fax:
34-5-4377413.
Sir, Azithromycin is an azalide antimicrobial agent that has been shown to concentrate and reach high intracellular concentrations in different types of human cells.1–3 It has been postulated that polymorphonuclear leucocytes (PMNs) can transport the drug to the site of infection. It also appears that fibroblasts may act as a reservoir for the drug in tissues allowing activity against organisms and possibly transferring azithromycin to phagocytic cells.4
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Correspondence Azithromycin has shown high intrinsic activity against Chlamydia trachomatis. In fact, a single oral dose of azithromycin is currently considered to be an alternative for the treatment of genital infections caused by this microorganism. Since C. trachomatis invades and multiplies within epithelial cells, the excellent response of C. trachomatis infections to azithromycin could be partially related to a potential capability of the drug to accumulate within these cells. The purpose of this study was to evaluate the intracellular penetration of azithromycin into tissue-cultured epithelial cells relative to that in human phagocytic cells. The tissue-cultured epithelial cell lines McCoy and Hep-2 (Flow Laboratories, Irvine, UK) were grown in supplemented minimal essential medium (Flow Laboratories) containing 10% fetal calf serum (Flow Laboratories) without antibiotics. Cells were detached from tissue culture bottles with trypsin–EDTA (Flow Laboratories), washed and suspended in Hanks’ balanced salt solution (HBSS) at a concentration of 5 106 cells per mL. PMNs were recovered from venous blood of healthy donors by dextran sedimentation and a Ficoll–Hypaque (Pharmacia Biotech, Uppsala, Sweden) gradient. Peritoneal macrophages were isolated from peritoneal effluents of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) as previously described.5 Cell preparations from CAPD donors always contained 75% peritoneal macrophages and 15% PMNs. Final cell suspensions were adjusted to 5 106 PMN, or peritoneal macrophages per mL in HBSS. Uptake of [14C]azithromycin (42.15 Ci/mg; Pfizer, Greton, CT, USA) by phagocytic and non-phagocytic cells was determined by the technique described by Pascual et al.5 Cells were incubated in HBSS containing different extracellular concentrations of azithromycin (0.125–10 mg/L). After different incubation periods at 37°C, cells were separated from the extracellular solution by centrifugation through a silicone oil barrier in a microcentrifuge tube. A 10 L aliquot of the extracellular medium and the entire cell pellet, obtained by cutting off the portion of the microcentrifuge tube containing the pellet, were placed in 3 mL of scintillation fluid (Ready Micro, Beckman Instruments, Inc., Beckman, Germany) and counted. After determination of the cell volume, the accumulation rate of azithromycin in cells (cellular to extracellular concentration ratio, C/E) was calculated as described previously. The intracellular penetration of azithromycin into phagocytic and non-phagocytic cells is shown in the Figure. Azithromcyin penetrated rapidly into both types of epithelial cells, reaching intracellular concentrations at least three times higher than the extracellular ones. At higher extracellular concentrations (10 mg/L) the intracellular concentrations of azithromycin in McCoy and Hep-2 cells were 35 6.2 and 58 10.8 mg/L respectively. This uptake was significantly lower than that
Figure. Kinetics of intracellular penetration of azithromycin in McCoy cells ( ), Hep-2 cells ( ), PMNs ( ) and peritoneal macrophages ( ). Experiments (n 5) were carried out at extracellular concentrations of 0.125 mg/L (A) and 1 mg/L (B). Data are expressed as the mean standard deviation. *P 0.05 relative to PMNs (analysis of variance).
observed in phagocytic cells. In fact, the C/E values of azithromycin in PMNs and peritoneal macrophages were greater than 23 and 60 respectively. At higher extracellular concentrations (10 mg/L), the intracellular concentrations of azithromycin in PMNs and peritoneal macrophages were 110 19 and 606 54 mg/L respectively. Azithromycin reaches high tissue concentrations at the site of infection as a result of PMN transportation and high intracellular concentrations in fibroblasts.4 There is little information, however, about the ability of this agent to concentrate within epithelial cells, the host cell for C. trachomatis. The penetration of azithromycin into tissue cultured human endometrial cells exceeded that of erythromycin by as much as eight-fold.1 This penetration rate was not affected by the presence of intracellular C. trachomatis. This study only evaluated high extracellular concentrations of azithromycin (10 mg/L) and C/E ratio values were not calculated. We have observed that even at very low extracelluar concentrations (0.125 mg/L) azithromycin concentrates several times within the cells used for susceptibility testing assays of C. trachomatis. It is interesting to note that the uptake of azithromycin by human peritoneal macrophages is much higher than that observed in PMNs. This may be an advantage over other macrolides in the treatment of infections due to susceptible organisms in patients undergoing CAPD.6 Nevertheless further studies are needed to elucidate this point. Azithromycin concentrates several times in tissuecultured epithelial cells. This phenomenon may partially explain the excellent response to this agent of genital infections caused by C. trachomatis.
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References 1. Raulston, J. E. (1994). Pharmacokinetics of azithromycin and erythromycin in human endometrial epithelial cells and in cells infected with Chlamydia trachomatis. Journal of Antimicrobial Chemotherapy 34, 765–76. 2. Pascual, A., Conejo, M. C., García, I. & Perea, E. J. (1995). Factors affecting the intracellular accumulation and activity of azithromycin. Journal of Antimicrobial Chemotherapy 35, 85–93. 3. Wildfeuer, A., Laufen, H. & Zimmermann, T. (1996). Uptake of azithromycin by various cells and its intracellular activity under in vivo conditions. Antimicrobial Agents and Chemotherapy 40, 75–9. 4. McDonald, P. J. & Pruul, H. (1991). Phagocyte uptake and transport of azithromycin. European Journal of Clinical Microbiology and Infectious Diseases 10, 828–33. 5. Pascual, A., Tsukayama, D., Kovarik, J., Gekker, G. & Peterson, P. (1987). Uptake and activity of rifapentine in human peritoneal macrophages and polymorphonuclear leukocytes. European Journal of Clinical Microbiology 6, 152–7. 6. Lam, Y. W., Flaherty, J. F., Yumena, L., Schoenfeld, P. Y. & Gambertoglio, J. G. (1995). Roxithromycin disposition in patients on continuous ambulatory peritoneal dialysis. Journal of Antimicrobial Chemotherapy 36, 157–63.
Decrease in serum levels of valproic acid during treatment with a new carbapenem, panipenem/betamipron J Antimicrob Chemother 1997; 39: 295–296 Kensuke Nagai*, Toko Shimizu, Akiko Togo, Mitsue Takeya, Yuko Yokomizo, Yasutaka Sakata, Toyojiro Matsuishi and Hirohisa Kato Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830, Japan *Tel:
81-942-35-3311; Fax:
81-942-38-1792.
Sir, Panipenem/betamipron, a new carbapenem, is a combined drug containing panipenem and betamipron, which has been released on the market in Japan. Panipenem is an antimicrobial agent and betamipron is an organic ion transport inhibitor for decreasing renal damage.1 We found low serum concentrations of valproic acid during treatment with panipenem/betamipron in three epileptic patients. The serum concentrations of valproic acid and other antiepileptic drugs were measured 2 h after oral administration by fluorescence polarization immunoassay in all patients. The serum concentrations of panipenem and betamipron were determined by highperformance liquid chromatography. Informed consent for the administration of panipenem/betamipron and for
taking blood and urinary samples for assays of drugs was obtained from patients. The first case was a 10-year-old boy who had been diagnosed as having pleural empyema. He was taking valproic acid (8.3 mg/kg every 8 h), carbamazepine and clonazepam. The antibiotic treatment was changed to iv panipenem/betamipron (20 mg/kg every 6 h) and clindamycin on the third day. His convulsions increased on the sixteenth day after panipenem/betamipron was started. The serum concentration of valproic acid was 70.7 mg/L before admission and decreased to 4.6 mg/L with an appropriate serum carbamazepine level of 6.7 mg/L at this time. The low level of valproic acid continued although the daily dose was increased Panipenem/ betamipron and clindamycin were continued because a clinical effect was observed and surgical treatment for pleural adhesions could not be done owing to the patient’s general condition. Panipenem/betamipron was administered for a total of 47 days. The serum levels of valproic acid were 25.8 mg/L and 45.8 mg/L on the third and eighth day respectively after stopping panipenem/ betamipron. The second case was an 8-year-old girl treated with a combination of valproic acid, carbamazepine, nitrazepam and phenytoin, who was admitted with recurrent pneumonia. The antibiotic treatment was changed to iv panipenem/betamipron (20 mg/kg every 8 h) on the tenth day, together with an iv antifungal agent, fluconazole. The serum level of valproic acid decreased after injection of panipenem/betamipron (Figure). The serum levels of panipenem and betamipron were 28.4 mg/L and 12.6 mg/L respectively at 1 h after injection, and 9.4 mg/L and 1.3 mg/L at 2 h, respectively. Panipenem/betamipron was discontinued on the 18th day because of clinical failure. The serum level of valproic acid returned to normal on the tenth day after stopping panipenem/betamipron. A 10-year-old girl with pleural empyema was the third case. She had been treated with valproic acid (5 mg/kg every 12 h) and carbamazepine. The antibiotic treatment was changed to iv panipenem/betamipron (20 mg/kg every 6 h) on the third day because she was not improving. Her convulsions increased in frequency on the second day after the start of the drug. The serum level of valproic acid was 26.5 mg/L after injection of panipenem/ betamipron. Valproic acid was substituted by phenobarbitone, which controlled her convulsions. Her clinical and laboratory findings were improved and panipenem/ betamipron was given for 53 days in total. The fractional excretion of valproic acid (the level of urinary valproic acid serum creatinine/ serum valproic acid urinary creatinine 100) was 0.06% at 0.5 h after the first injection of panipenem/betamipron and was increased to 0.21% at 18 h. The carbamazepine levels before and during panipenem/betamipron treatment were 9.0 mg/L and 9.2 mg/L, respectively. The mechanisms of pharmacological interaction
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Figure. Correlation of administration between panipenem/betamipron and valproic acid in case 2.
between panipenem/betamipron and valproic acid together with administration of carbamazepine were not clearly explained in our cases. The binding rates of valproic acid to serum protein vary between 80% and 94% in both healthy subjects and patients with epilepsy,2 and those of panipenem and betamipron are 2.5% to 11.5% and 70%,3 respectively. The competition of valproic acid and panipenem/betamipron for binding proteins might be considered. It is possible that panipenem/betamipron might accelerate the renal excretion of valproic acid because the fractional excretion of valproic acid increased after panipenem/betamipron was given in the third case. Interactions between the two drugs could be associated with the administration of carbamazepine because it was also given to all three patients and accelerates the metabolism of valproic acid in the liver;2 however, no change in the serum carbamazepine level was found. Further studies in an experimental model are required to determine the interaction between panipenem/
betamipron and valproic acid with carbamazepine. The monitoring of serum valproic acid levels is desirable in patients concurrently being treated not only with panipenem/betamipron but with other carbapenems.
References 1. Shimada, K. (1994). New antimicrobial agent series XLVI: panipenem/betamipron. Japanese Journal of Antibiotics 47, 219–44. 2. Rall, T. W. & Schleifer, L. S. (1990). Drugs effective in the therapy of the epilepsies. In Goodman and Gilman’s The Pharmaco logical Basis of Therapeutics, 8th edn (Gilman, A. G., Rall, T. W., Nies, A. S. & Taylor, P., Eds), pp. 436–62. Pergamon Press, New York, NY. 3. Nakashima, M., Uematsu, T., Kanamaru, M., Tajima, M., Naganuma, H., Ichikawa, M. et al. (1991). Phase I study of panipenem/betamipron II—multiple dose study. Chemotherapy (Tokyo) 39, Suppl. 3, 265–88.
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