Hospital use of systemic antifungal drugs: a multi-center surveillance update from Germany.

BACKGROUND: The consumption of antifungal agents increased over the last decade, resulting in the development of resistant organisms and causing a significant pharmaco economic burden. Antifungal drugs are widely used for the treatment of systemic fungal infections and high-risk patients, especially with severe hematological or oncological conditions. Up to date, there are no reliable and systematically reported data on the consumption of antifungal substances on a nationwide level available. The presented study gives an update to the previously published multicenter study investigating antifungal consumption in different settings from five university hospital centers in Germany from 2001 to 2003. METHODS: Consumption data for systemic antifungal drugs were obtained through the hospital pharmacies for 2001-2003 and 2008-2011 regarding the medical and surgical services of five university hospital centers in Germany (A-E). Drug use densities were calculated as yearly RDDs/100 patient days. These calculations were performed for the surgical and medical services, and independently for surgical and medical ICUs, as well as for the hematology-oncology services. RESULTS: We report an increased utilization of systemic antifungal drugs in both study periods. The mean drug use density (mean value of all 5 hospitals) in the medical services increased by 24% between 2001 and 2003. In 2011, this value was 37% above the level from 2001 (12.4 RDD/100 patient days in 2001, 15.4 RDD/100 patient days in 2003, 17.0 RDD/100 patient days in 2011). The 4-year average drug use density (2008-2011) of medical services ranged between 11.6 RDD/100 patient days (hospital E) and 23.8 RDD/100 patient days (hospital A). Drug use densities were in medical intensive care units 29.4 RDD/100 patient days and hematology-oncology services 49.9 RDD/100 patient days. CONCLUSIONS: Despite the variability of the prescribing patterns between the tertiary hospitals, the presented pharmaco-epidemiological data are a cornerstone for the initiation and implementation of effective antifungal stewardship programmes and might serve as important benchmarking information for other hospitals with similar structures and baseline settings.

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery.

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.

Visualization of sialyl Lewis(X) glycosphingolipid microdomains in model membranes as selectin recognition motifs using a fluorescence label.

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the cellular immune response. For efficient recognition, the relevant carbohydrate epitope sialyl Lewis(X) (sLe(X); alpha-Neup5Ac-(2-->3)-beta-Galp-(1-->4)-[alpha-Fucp-(1-->3)]GlcpNAc) has to be arranged in clusters. We describe the synthesis of the sLe(X)-glycosphingolipid (sLe(X)-GSL) with a NBD fluorescence label in the tail region, which allows the direct visualization of sLe(X)-GSL microdomains to very low concentrations (0.01mol%) in various planar phosphocholine matrices by fluorescence microscopy. Cell rolling experiments of E-selectin expressing cells along these membranes confirmed that the fluorescence analog behaves similar to the naturally occuring sLe(X)-GSL. This is direct evidence for recent hypotheses on multivalent sLe(X) binding as molecular basis for selectin-mediated cell rolling.

Biosensor-based evaluation of liposomal behavior in the target binding process.

In this study we evaluated the quartz crystal microbalance (QCM) as a biosensor for a real-time investigation of liposomal binding, under dynamic flow conditions, onto target proteins immobilized at the sensor. The mass-sensitive frequency changes of quartz sensors allow for a quantification of the liposomal binding process. Furthermore, simultaneous damping analysis gives an insight into liposomal behavior, such as the degree of liposomal deformation or spreading at the target surface. In this study a series of liposomes was evaluated, differing in the kind and concentration of ligands interacting with appropriate target proteins. It became evident that an increase in homing device concentration accelerated deformation and flattening of liposomes, triggering a fusion process. Furthermore, liposomal deformation corresponded with the binding affinity of target molecules, comparing biotin/avidin with E-selectin/ligand interactions. Deformation could be emphasized using dioleoylphosphatidylethanolamine (DOPE) as a fusiogenic membrane component, while sterical stabilization by polyethylenglycol (PEG-PE) appeared in a low degree of deformation. Consequently, the online detection of liposomal target binding by QCM is an excellent facility to control and predict the liposomal behavior at the target site for increasing therapeutic potency.

Nanoporous aluminum oxide membranes for filtration and biofunctionalization.

Nanoporous aluminum oxide membranes with high open porosity are prepared by anodic oxidation. Conventional self-supporting as well as mechanically stabilized nanoporous membranes are produced from aluminum plates and microimprinted aluminum foils, respectively. The mechanically stabilized membranes are characterized by very thin membrane parts stabilized by surrounding thick bridges. The minimal thickness of these thin membranes with open pores on both sides is 1 microm, with a mean pore size of the parallel open pores of 185 nm. With these two kinds of membrane the flow rates for cross filtration can be tuned over a wide range. With the mechanically stabilized membranes, substantially higher flow rates are achieved and experiments that cannot be performed with thicker membranes become possible. The biofunctionalization of the pore walls with archaebacterial tetraether lipids is realized and proved using aminated semiconductor nanocrystals. The lipid layer deposited on the pore walls also changes the filtration properties.

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes.

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 microg/micromol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.

The detection of UV-induced membrane damages by a combination of two biosensor techniques.

Ketoprofen is an important anti-inflammatory drug, but its dermal application is limited because of the photosensitizing properties causing phototoxic reactions of the skin when exposed to UV light. We have recently investigated the peroxide formation of ketoprofen in solutions of linoleic acid during UV irradiation. To continue these studies and focus on UV-induced changes in membrane integrity and barrier function we established an in vitro model using two biosensor techniques simultaneously. Support-fixed bilayers were irradiated with different doses of UV-B up to damaging intensities with or without ketoprofen. Cyclic voltammetry was carried out to detect alterations in membrane permeability; quartz crystal microbalance (QCM) measurements were helpful in analyzing whether a permeability increase was caused by depletion of membrane components. In absence of ketoprofen, increasing UV-B doses induce membrane permeabilities of both unsaturated and saturated bilayers. QCM measurements could not reveal a significant loss of membrane components as a reason for the permeability. In contrast, 0.3 mM ketoprofen induced a dose-dependent increase in membrane permeability. QCM results indicated a mass loss. Although this model does not explain all molecular mechanisms of membrane damage by ketoprofen, the combined application of both QCM and CV is a novel and powerful tool to investigate functional mechanisms of UV-induced membrane damages.

Hospital use of systemic antifungal drugs.

BACKGROUND: Sales data indicate a major increase in the prescription of antifungal drugs in the last two decades. Many new agents for systemic use that only recently have become available are likely to be prescribed intensively in acute care hospitals. Sales data do not adequately describe the developments of drug use density. Given the concerns about the potential emergence of antifungal drug resistance, data on drug use density, however, may be valuable and are needed for analyses of the relationship between drug use and antifungal resistance. METHODS: Hospital pharmacy records for the years 2001 to 2003 were evaluated, and the number of prescribed daily doses (PDD, defined according to locally used doses) per 100 patient days were calculated to compare systemic antifungal drug use density in different medical and surgical service areas between five state university hospitals. RESULTS: The 3-year averages in recent antifungal drug use for the five hospitals ranged between 8.6 and 29.3 PDD/100 patient days in the medical services (including subspecialties and intensive care), and between 1.1 and 4.0 PDD/100 patient days in the surgical services, respectively. In all five hospitals, systemic antifungal drug use was higher in the hematology-oncology service areas (mean, 48.4, range, 24 to 101 PDD/100 patient days, data for the year 2003) than in the medical intensive care units (mean, 18.3, range, 10 to 33 PDD/100) or in the surgical intensive care units (mean, 10.7, range, 6 to 18 PDD/100). Fluconazole was the most prescribed antifungal drug in all areas. In 2003, amphotericin B consumption had declined to 3 PDD/100 in the hematology-oncology areas while voriconazole use had increased to 10 PDD/100 in 2003. CONCLUSION: Hematology-oncology services are intense antifungal drug prescribing areas. Fluconazole and other azol antifungal drugs are the most prescribed drugs in all patient care areas while amphotericin B use has considerably decreased. The data may be useful as a benchmark for focused interventions to improve prescribing quality.

Antibiotic use in German university hospitals 1998-2000 (Project INTERUNI-II).

In a pilot study we established a hospital antibiotic use database from which estimates for antibiotic consumption in German hospitals (expressed as defined daily doses per 100 patient days, DDD/100) can be retrieved for both benchmarking and antibiotic use and resistance research purposes. Data from eight university hospitals (1998-2000) showed a mean antibiotic use density of 60.1 DDD/100 in the surgical and 79.3 DDD/100 in the medical services. Antibiotic use was higher in the intensive care units (surgery: 146 DDD/100, medicine: 187 DDD/100) than in haematology-oncology services (110.8 DDD/100) or in other surgical (51.6 DDD/100) and medical (66 DDD/100) service areas. There were major differences in the use of specific antibacterial drug classes between service areas. The established database allows detailed analysis in antibacterial drug use and can be linked to bacterial resistance surveillance databases.

Sensing specific adhesion of liposomal and micellar systems with attached carbohydrate recognition structures at lectin surfaces.

A quartz crystal microbalance was used to investigate the adsorption behavior of liposomes and mixed micelles with attached carbohydrate recognition structures at lectin-coated quartz plates. With a self-assembly technique, the quartz was coated with the lectin Concanavalin A. In a first attempt, liposomes of natural soybean PC as well as synthetic POPC, containing 10% reactive N-Glut-PE each, were decorated with a mannopyranoside recognition structure to investigate the specific adsorption at the lectin-coated quartz surface in dependence on the concentration. In a second model, the bile salt sodium cholate was introduced to solubilize the mannopyranoside-modified liposomes and to transform them into mannopyranoside-modified binary mixed micelles. The adsorption of these micelles was further investigated. In a third approach, the adsorption behavior of mannopyranoside-modified ternary mixed bile salt-phosphatidylcholine-fatty acid micelles was characterized with sodium laurate, palmitate, and oleate as fatty acids. The micelles with oleate showed only a small frequency decrease, whereas the micelles with laurate and palmitate induced higher frequency changes. A dependence on the alkyl chain length could be detected. While the adsorption of liposomes containing recognition structures at QCM surfaces is nowadays well-established, the QCM detection of the adsorption of mixed bile salt micelles, transformed from these liposomes by solubilization, is a novel and very promising field for the development of innovative colloidal drug delivery systems.

[Antibiotic Use at German University Hospitals (Project INTERUNI-II). Results for Medical Intensive Care, Hematology-Oncology, and Other Medical Service Areas]. / Antibiotikaanwendung an deutschen Hochschulkliniken (Projekt INTERUNI-II). Ergebnisse für medizinische Kliniken unter Berücksichtigung von Intensivpflegestationen, onkologischen Stationen und sonstigen Pflegebereichen.

BACKGROUND: Excessive antibiotic use increases the risk of development and dissemination of bacterial resistance. A comparative analysis of the correlation between hospital antibiotic consumption and rates of bacterial resistance is needed for a better understanding of the complex relationship between antibiotic use and resistance. Apart from economic and market research studies, estimates of antibiotic consumption in German hospitals, however, are not available. METHODS: In a pilot project (INTERUNI-II), retrospective data from eight university hospital pharmacies covering the period 1998, 1999, and 2000 were collected to obtain estimates for the antibiotic use densities in the medical services of teaching hospitals. Antibiotic use densities were expressed as prescribed daily doses per 100 occupied bed days (PDD/100). The definition of prescribed daily doses was according to guidelines for antimicrobial therapy in adults with normal renal and hepatic function used in the participant hospitals. Means and ranges of antibiotic use densities were separately assessed for medical intensive care units (MICU), hematology-oncology services (HEMONC), and other medical services (OTHER MED). RESULTS: Mean antibiotic use density in internal medicine was 55.2 PDD/100 overall, ranging between 39.4 and 75.8 PDD/100 in the eight participant hospitals. In seven hospitals antibiotic use density increased during the years of observation. Antibiotic use was higher in MICU areas (3-year average, 122.3; range, 98-167 PDD/100) than in HEMONC (3-year average, 86.9; range, 67.8-129.4 PDD/100) and OTHER MED areas (3-year average, 42.8; range, 31.7-50.6 PDD/100). There was an increasing use of oral antibiotics resulting in a substantial proportion of oral agents among all antibacterial drugs outside MICU areas (year 2000, HEMONC, range, 36-74% of all PDD; OTHER MED, range, 43-59% of all PDD). beta-lactam antibiotics were the most frequently prescribed drugs (3-year average, 22.6 PDD/100). 56% of beta-lactam PDD belonged to the class of broad-spectrum beta-lactams (ranges, MICU, 49-82%; HEMONC, 61-89%; OTHER MED, 24-58%). Fluoroquinolones were the second most prescribed drug class (3-year average, 13 PDD/100). They were most frequently used in HEMONC (3-year average, MICU, 14.5; HEMONC 26.5; and OTHER MED 8.6 PDD/100, respectively). There was considerable variation between participant hospitals in the use of specific drug classes in given patient care areas. CONCLUSION: This retrospective study showed significant variation in overall and specific antibacterial drug class use between German teaching hospital medical services and defined patient care areas. Given the variation in the obtained estimates, targeted prospective hospital antibiotic use surveillance with fast data acquisition and analysis might offer an excellent opportunity to evaluate the impact of differences in antibiotic use and of revised therapy guidelines on the evolution of nosocomial bacterial resistance.

Stability of monomolecular films of archaebacterial tetraether lipids on silicon wafers: a comparison of physisorbed and chemisorbed monolayers.

The monomolecular organisation of symmetric, chemically modified tetraether lipids caldarchaeol-PO(4) was studied using Langmuir film balance, ellipsometry, and atomic force microscopy (AFM). Solid silicon wafer substrates were modified to hydrophobic, hydrophilic, and amino-silanised surfaces; and Langmuir-Blodgett (LB)-films were transferred onto each. LB-caldarchaeol-PO(4) films were subjected to further rinsing with organic solvent and additional physical treatments, to compare their resistance and stability on chemisorbed (amino-silanised) and physisorbed (hydrophobic and hydrophilic) surfaces. The resistance and stability of these monolayer films was characterized by ellipsometry and AFM, and film thickness was determined using ellipsometry. AFM was also employed to observe surface morphology. Monolayer films on hydrophobic surfaces were found to be more resistant to rinsing with organic solvent and additional physical treatments than monolayer films on either amino-silanised or hydrophilic surfaces. The hydrophobic effect with hydrophobic surfaces appears to support the formation of stronger caldarchaeol-PO(4) films on silicon wafer substrates, with increased resistance and stability.

AFM and ellipsometric studies on LB films of natural asymmetric and symmetric bolaamphiphilic archaebacterial tetraether lipids on silicon wafers.

The monomolecular organisation of the natural asymmetric and symmetric bolaamphiphilic (archaebacterial tetraether lipids) was studied using a combination of Langmuir film balance, ellipsometry, and atomic force microscopy (AFM). Monolayer films were transferred onto silicon wafer substrates. After transfer of the lipids onto several kinds of silicon wafer substrates (hydrophobic, hydrophilic, and amino-silanised), the monolayer films were further investigated by ellipsometry and AFM in order to determined whether bolaamphiphiles are oriented differently at the water-air interface, including "horseshoe" and "upright" configurations. The thickness of the monolayer film, which was determined by ellipsometry, represents a combined mean value of the domains. The surface morphology, which was evaluated by AFM showed that in all films, the domains were arranged in small domains or the most homogeneous organisation, depending on the properties of the wafer silicon substrates. From all films, the hydrophobically transferred lipids showed the most homogeneous organisation on the substrates. After transfer onto hydrophilic and amino-silanised surfaces, the lipids were arranged in small islands on the substrates.

Surface modification of medical-grade polyurethane by cyanurchloride-activated tetraether lipid (a new approach for bacterial antiadhesion).

The surface of high-grade medical polyurethane was chemically modified and coated with tetraether lipid, employing cyanurchloride as coupling agent. The surfaces were initially grafted with hexamethylene diisocyanate and subsequently treated with water or hexamethylene diamine to generate free amino groups on the surface. This provides a convenient way for covalent coupling of tetraether lipids. These lipid-coated surfaces were investigated for bacterial adhesion using Pseudomonas aeruginosa. All lipid-coated surfaces significantly reduced bacterial adhesion. Surface topology and individual modification steps were controlled by contact angle measurements, attenuated total reflectance infrared spectroscopy, X-ray photoelectron spectroscopy, confocal laser scanning microscopy, and environmental scanning electron microscopy. This new surface modification approach may reduce the adhesion of bacteria on biomaterials.

Lessons learned from clustering of fluorinated glycolipids on selectin ligand function in cell rolling.

Selectin-induced leukocyte rolling along the endothelium is an essential step in the cellular immune response. Since clustering of binding epitopes is thought to be crucial for selectin-ligand interaction, we focused on requirements of ligand clusters in a flow chamber study. Neoglycolipids bearing the binding epitope sialyl Lewis X (sLeX) were used as artificial ligands in model membranes. sLeX ligands or matrix lipids or both were applied with partially fluorinated alkyl chains to increase the ligand cluster separation tendency. Cluster size, their inner structure, and separation distance were evaluated with high resolution by scanning force microscopy (SFM) and correlated with binding or rolling of E-selectin-expressing cells. Fluorination of only one component, sLeX ligand or matrix lipid, leads to a very high separation tendency and impeded cell rolling, although firm cell adhesion could be observed down to 0.005 mol % ligand concentration. As a sign of total immiscibility, cluster size increased with ligand concentration, and resulting excessive ligand densities within the clusters prevent cell rolling. Fluorination of both sLeX ligands and matrix created small clusters which could serve as rolling patches. Our results confirmed that cluster size and separation distance controlled by a certain miscibility of ligand and matrix as well as a sufficiently diluted ligand concentration within the clusters are necessary for cell rolling. Within this work, selectin ligand clustering and its ability to mediate cell rolling are presented as a balance between multivalency of binding and sufficient flexibility of the single epitopes. This might be helpful for better understanding the function of the natural selectin ligands.

Cooperation between lateral ligand mobility and accessibility for receptor recognition in selectin-induced cell rolling.

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the immune response. Several in vitro studies showed that this cell rolling is a highly regulated adhesion phenomenon, controlled by the kinetics and forces of selectin-ligand interactions. In the flow chamber study presented here, we focused on the requirements on the ligand structure in this context. A series of neoglycolipids bearing the binding epitope Sialyl Lewis X was synthesized and used as artificial ligands. These lipids differed in their spacer structures between headgroup and membrane anchor, resulting in a gradual variation in accessibility and mobility of the binding epitope when immobilized in model membranes. Consequently, analysis of cell rolling along such membranes allowed correlation of ligand structures and functionality. All model membranes containing such ligands were further characterized by film balance measurements, epifluorescence, and atomic force microscopy. Generally, the glycolipids exhibited a high tendency for lateral aggregation, but the resulting clusters were of different morphology. This was also reflected by strong differences in the rolling experiments. Our results confirm that, in addition to a sufficient headgroup accessibility, the cell rolling process is governed by two further interdependent factors: (i) the headgroup flexibility caused by the intramolecular uncoupling between the headgroup and the hydrophobic moiety due to introduction of a spacer, and (ii) the stiffness of the molecules resulting from their supramolecular arrangement in clustered assemblies. Since both factors are influenced simultaneously by the spacer modification, we present for the first time a clear correlation between structural aspects of selectin ligands and their ability to mediate cell rolling. This might help to develop a better understanding for the function of the natural selectin ligands.

Functional microdomains of glycolipids with partially fluorinated membrane anchors: impact on cell adhesion.

Functional microdomains of glycolipids were designed by mixing neoglycolipids with partially fluorinated alkyl (F-alkyl) chains and matrix lipids with alkyl chains. Fluorescence images of the mixed lipid monolayers at the air-water interface demonstrated that it is possible to control both size and distribution of the microdomains by means of the strong demixing of alkyl and F-alkyl membrane anchors, while the carbohydrate head groups seemed to play a rather minor role. These microdomains in monolayers could be transferred onto hydrophobized substrates and subjected to experiments in a dynamic flow chamber. The results obtained here clearly indicated that the dynamic adhesion of Chinese hamster ovarial cells expressing E-selectin (CHO-E cells) on a lipid monolayer containing microdomains of sialyl Lewisx (sLex) can be both enhanced and reduced by controlled demixing of ligands and matrices. Moreover, the same clusters of sLex could also be formed in giant lipid vesicles, which can be used as a model cell that locally expresses biospecific functions.

Update on glycopeptide use in german university hospitals.

BACKGROUND: A previous study has shown considerable variation in glycopeptide use from 1992 through 1994 among four university hospitals in southern Germany. Active antimicrobial management in one of the hospitals was associated with the containment of glycopeptide consumption in the medical and surgical service at < 1.5 defined daily doses (DDD)/100 patient days in the subsequent period. In the present study, more recent data on comparative glycopeptide use in German university hospitals were analyzed. MATERIALS AND METHODS: Hospital pharmacy records from 1998 through 2000 were evaluated. The number of DDD (definition according to the World Health Organization [WHO]/Anatomic and Therapeutic Classification [ATC] index) per 100 patient days was calculated to compare glycopeptide use in different medical and surgical service areas between eight German university hospitals. RESULTS: The 3-year averages in recent glycopeptide use for the eight hospitals ranged between 1.3 and 8.8 DDD/100 patient days in the medical services, and between 0.7 and 1.8 DDD/100 patient days in the surgical services. Only one of the eight hospitals showed medical service glycopeptide use of < 1.5 DDD/100 patient days. In most hospitals, glycopeptide use was higher in the medical intensive care units (ICU) (median 8.6; range 4.3 to 12 DDD/100 patient days, data for the year 2000) than in the surgical ICUs (median 6.7; range 1.2 to 8.6 DDD/100 patient days, data for the year 2000). High use was also observed for hematology-oncology services (median 7.5; range 2.7 to 15.7 DDD/100 patient days, data for the year 2000). CONCLUSION: These recent data from a larger hospital sample confirm large variations in glycopeptide use, identify hematology-oncology services as a significant prescribing source along with ICUs, and indicate areas of probable overuse of glycopeptide antibiotics. The data may be useful as a benchmark for further focused drug use control interventions.