Michael V. Schintler

Soft tissue and bone penetration abilities of daptomycin in diabetic patients with bacterial foot infections

OBJECTIVES In the attempt to overcome increasing glycopeptide- and methicillin-resistant soft tissue infections, daptomycin is presently considered as an attractive alternative to the class of glycopeptides. However, daptomycin dosing and its ability to penetrate into inflamed target tissues are still a matter of controversy. Thus, in the present investigation, we set out to evaluate daptomycins ability to penetrate into inflamed subcutaneous adipose tissue and bone in diabetic patients presenting with severe bacterial foot infection. PATIENTS AND METHODS The microdialysis technique was utilized to collect interstitial space fluid from inflamed subcutaneous adipose tissue and metatarsal bone. Plasma and unaffected subcutaneous adipose tissue served as reference compartments. Serial sampling of specimens at steady-state was performed from 0 to 8 h post-dose in five patients (Group A) and from 8 to 16 h after study drug administration in another group of four patients (Group B). In all subjects, daptomycin was administered intravenously once daily at a dosage of 6 mg/kg body weight for 4 consecutive days at minimum. RESULTS Equilibrium between free tissue and plasma concentrations was achieved approximately 2 h post-infusion. Under steady-state conditions, the degree of tissue penetration was assessed by the calculation of the ratio of free (f) AUC of daptomycin in plasma to the fAUC in tissues. The mean ratios of the fAUC0-16 tissue to the fAUC0-16 plasma were 1.44, 0.98 and 1.08 for healthy tissue, inflamed subcutaneous adipose tissue and bone, respectively. The corresponding ratios of the fAUCs from 0 to 24 h were 1.54, 1.06 and 1.17, respectively. CONCLUSIONS With the reservation that pharmacokinetic-pharmacodynamic targets for daptomycin in tissues are currently not established, we conclude that daptomycin given at intravenous doses of 6 mg/kg body weight once daily may be considered an effective treatment regimen in diabetic patients suffering from bacterial foot infection and osteomyelitis.

High fosfomycin concentrations in bone and peripheral soft tissue in diabetic patients presenting with bacterial foot infection

OBJECTIVES Appropriate antimicrobial therapy and surgical intervention may be required in diabetic patients presenting with severe bacterial foot infection. Methicillin-resistant Staphylococcus aureus (MRSA) agents such as fosfomycin are increasingly in demand because of recent concern regarding vancomycin and daptomycin efficacy and constant use. Intravenous fosfomycin is approved for the therapy of severe soft tissue infections and is highly active against methicillin-susceptible S. aureus and MRSA. in the present study we investigated fosfomycins ability to penetrate bone tissue in diabetic patients suffering from severe bacterial foot infection. PATIENTS AND METHODS The well established microdialysis technique was utilized to determine fosfomycin concentrations in metatarsal bone in nine patients scheduled for partial bone resection due to bacterial foot infection and osteomyelitis. Plasma and unaffected subcutaneous adipose tissue served as reference compartments. RESULTS After a single intravenous dose of approximately 100 mg of fosfomycin per kg of body weight, the mean C(max), T(max) and AUC(0-6) for bone were 96.4 mg/L, 3.9 h and 330.0 mg x h/L, respectively. The degree of tissue penetration as determined by the ratios of the AUC(0-6) for bone to plasma and for subcutaneous adipose tissue to plasma were 0.43 +/- 0.04 and 0.76 +/- 0.05, respectively. CONCLUSIONS On the basis of relevant pharmacokinetic-pharmacodynamic indices, it seems that fosfomycin is an effective antibiotic for the treatment of deep-seated diabetic foot infections with osseous matrix involvement.

Linezolid concentrations in infected soft tissue and bone following repetitive doses in diabetic patients with bacterial foot infections

The present study aimed at assessing unbound extracellular concentrations of linezolid in inflamed soft tissue and bone of diabetic patients suffering from severe bacterial foot infections. Linezolid was administered intravenously twice daily at a dosage of 600 mg. At steady-state conditions, the microdialysis technique was utilised to sample serially interstitial space fluid from inflamed subcutaneous adipose tissue and metatarsal bone from 0-8h post dose in three representative patients. Mean peak concentrations of free linezolid in plasma, healthy subcutis, inflamed subcutis and cancellous bone were 16.6+/-3.0, 15.5+/-2.5, 15.8+/-2.8 and 15.1+/-4.1mg/L, respectively. The degree of tissue penetration as expressed by the ratio of the area under the concentration-time curve of free linezolid from 0-12h (fAUC(0-12)) in tissue to the fAUC(0-12) in plasma was 1.32+/-0.09, 1.12+/-0.22 and 1.09+/-0.11 for healthy subcutis, inflamed subcutis and bone, respectively. Based on currently available pharmacokinetic/pharmacodynamic targets, we conclude that linezolid administered at 600 mg twice daily may be considered an effective treatment in diabetic patients suffering from bacterial foot infection complicated by osteomyelitis.

Novel peptidoglycan-based diagnostic devices for detection of wound infection

Detection of wound infection is based on evaluation of the well-known signs of inflammation like rubor (redness), calor (heat), tumor (swelling), and dolor (pain) by medical doctors and/or time-consuming procedures requiring special machinery. There is currently no rapid diagnostic device available for the indication of wound infection, which would especially be helpful in home care of chronic ulcer patients. In this study, a new concept for a fast diagnostic tool for wound infection based on lysozyme and elastase triggered release of dye from a peptidoglycan matrix was investigated. The matrix consisted of alginate/agarose and peptidoglycan covalently labeled with Remazol brilliant blue. Lysozyme activity in postoperative wounds and decubitus wound fluids was significantly elevated upon infection (4830 ± 1848 U mL(-1)) compared to noninfected wounds (376 ± 240 U mL(-1)). Consequently, incubation of 8% (w/v) labeled agarose/peptidoglycan blend layers with infected wound fluid samples for 2 h at 37 °C resulted in a 4-fold higher amount of dye released than measured for noninfected wounds. For alginate/peptidoglycan beads, a 7-fold higher amount of dye was released in case of infected wound fluid samples compared to noninfected ones. Apart from lysozyme, proteases [i.e., gelatinase matrix metalloproteinase MMP-2 and MMP-9 and elastase] were detected in wound fluids (e.g., using Western blotting). When dosed in ratios typical for wounds, a slight synergistic effect was measured for peptidoglycan hydrolysis (i.e., dye release) between lysozyme and these proteases. Incubation of a double-layer system consisting of stained and nonstained peptidoglycan with infected wound fluids resulted in a color change from yellow to blue, thus allowing simple visual detection of wound infection.

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid

Abstract:  Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non‐infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non‐infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.

A Role for Rab7 in the Movement of Secretory Granules in Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP‐bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over‐expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus‐end motor, dynein.

Analysis of myeloperoxidase activity in wound fluids as a marker of infection

Background Neutrophilic polymorphonuclear leukocytes play a crucial role in the host defence against bacterial and fungal infections. They participate in the inflammatory response through the liberation of peptides and enzymes like myeloperoxidase (MPO). Therefore, MPO has a potential as a marker enzyme for the diagnosis of wound infection. Methods Substrate specificities and reaction pathways of MPO were investigated for new MPO substrates: crystal violet, leuco crystal violet, fast blue RR (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt) and various systematically substituted model substrates based on 2,7-dihydroxy-1-(4-hydroxyphenylazo)naphtalene-3,6-disulphonic acid. In addition, fast blue RR was covalently bound to siloxanes allowing immobilization of the substrate, while cellobiosedehydrogenase was integrated for generation of hydrogen peroxide required by MPO. Results Elevated concentrations of MPO were found in infected wounds compared with non-infected wounds (92.2 ± 45.0 versus 1.9 ± 1.8 U/mL). Various soluble and immobilized substrates were oxidized by MPO in wound samples and the influence of substrate structure and reaction pathways were elucidated for selected compounds. Conclusions Incubation of different MPO substrates with infected wound fluid samples resulted in a clear colour change in the case of elevated MPO concentrations, thus allowing early diagnosis of wound infection.

Management of an unfortunate triad after breast reconstruction: Pyoderma gangrenosum, full-thickness chest wall defect and Acinetobacter Baumannii Infection

If diagnosed late, post-surgical pyoderma gangrenosum (PSPG) is a rare, tricky and potentially life-threatening complication. Once diagnosed, immunosuppressive agents may provoke further complications. Well-intentioned extensive serial debridement may cause deep skin and soft-tissue defects, requiring skin grafting and possible flap surgery. The combination of necessary immunosuppressive treatment, protracted hospital stay and broad-spectrum systemic antimicrobial therapy may encourage serious acquired multidrug resistance (MDR). We report an unfortunate triad following breast reconstruction of PSPG, full-thickness chest wall defect and MDR with Acinetobacter baumannii infection. Interdisciplinary treatment using free flap surgery and negative-pressure wound therapy with instillation therapy (V.A.C.Instill(R) Wound Therapy) enabled survival and complete wound closure.

Accidental perforating bone injury using the EpiPen® autoinjection device

Epinephrine is the drug of choice for the treatment of acute anaphylactic reactions (1). EpiPen (DEY, Napa, CA, USA) is widely prescribed by dermatologists and allergists for self-treatment of patients with lifethreatening allergic reactions (2). The pen works as an autoinjector device and contains 0.3 mg of epinephrine. Simultaneous accidental injections into fingers and palmar surface have been observed upon improper use. Until now, perforating bone injuries using an autoinjection device have not been reported in the literature. Because of its rapid effect on the important pathophysiologic events in an anaphylactic reaction, this drug is recommended for self-application to patients at risk from anaphylactic reactions, such as individuals with a history of anaphylaxis following hymenopteran stings or ingestion of certain foods (3). EpiPen is a disposable drug delivery system with a spring-activated concealed needle intended for intramuscular application. The EpiPen is designed as an emergency supportive therapy only and is not a substitute for immediate medical care. Initially, this autoinjector design was developed for the military to deliver antidotes in the event of a poison gas attack. It was ideal because of the need for a fast, convenient method of giving life-saving medication in a high-stress situation. Subsequently, the same autoinjector system was used with other drugs as part of the NASA Manned Space Flight Program. The EpiPen autoinjector resulted from the realization that patients experiencing serious allergic reactions may also be fearful of injecting themselves with life-saving medication or incapable of using a conventional syringe. We report about a 37-year-old woman who suffered an accidental autoinjection injury. While preparing the EpiPen autoinjection device for her mother, who got stung by an insect, she sustained a perforating injury of the index finger, with the needle entering the palmar side, going through the distal phalanx, and exiting through the dorsal side. On clinical examination, the index finger presented a rose color and appeared to be well perfused and warm. We found two tiny puncture wounds, one on the palmar side of the fingertip and the other one dorsal, proximal to the nail. There were no signs of ischemia. The patient developed pain after applying pressure on her index fingertip, but reported no loss of sensation. To exclude any inoculation of a foreign body in the fingertip, an X-ray was performed. We could not find any foreign body but discovered discrete signs of bone perforation in the distal phalanx in the anterior–posterior as well as in lateral view (Fig. 1). The patient was concerned about the needle having caused an inoculation of AL LERGY 2 0 0 5 : 6 0 : 2 5 9 – 2 6 7 • COPYRIGHT a 2005 BLACKWELL MUNKSGAARD • ALL R IGHTS RESERVED • CONTRIBUT IONS TO THIS SECT ION WILL NOT UNDERGO PEER REVIEW, BUT WILL BE REV IEWED BY THE ASSOCIATE EDITORS •

Novel protease-based diagnostic devices for detection of wound infection

A gelatinase‐based device for fast detection of wound infection was developed. Collective gelatinolytic activity in infected wounds was 23 times higher (p ≤ 0.001) than in noninfected wounds and blisters according to the clinical and microbiological description of the wounds. Enzyme activities of critical wounds showed 12‐fold elevated enzyme activities compared with noninfected wounds and blisters. Upon incubation of gelatin‐based devices with infected wound fluids, an incubation time of 30 minutes led to a clearly visible dye release. A 32‐fold color increase was measured after 60 minutes. Both matrix metalloproteinases and elastases contributed to collective gelatinolytic enzyme activity as shown by zymography and inhibition experiments. The metalloproteinase inhibitor 1,10‐phenanthroline (targeting matrix metalloproteinases) and the serine protease inhibitor phenylmethlysulfonyl fluoride (targeting human neutrophil elastase) inhibited gelatinolytic activity in infected wound fluid samples by 11–37% and 60–95%, respectively. Staphylococcus aureus and Pseudomonas aeruginosa, both known for gelatinase production, were isolated in infected wound samples.