Intravenous Cefazolin Achieves Sustained High Interstitial Concentrations in Open Lower Extremity Fractures.

BACKGROUND: Infection remains a serious clinical concern in patients with open fractures, despite timely antibiotic administration and surgical debridement. Soft tissue and periosteal stripping may alter local tissue homeostasis and antibiotic pharmacokinetics in the injured limb. The tissue (interstitial) concentration of intravenously administered antibiotics at an open fracture site has not been characterized using direct sampling techniques. QUESTION/PURPOSE: We performed this study to evaluate the concentration and pharmacokinetics of intravenously delivered cefazolin at an open fracture site after surgical debridement. METHODS: Twelve patients with an open fracture distal to the knee who presented at a regional Level I trauma center were approached for enrollment in this nonrandomized, observational study. Of the 12 patients, eight adults (one female, seven male) with a median age of 32 years (range 23 to 51 years) were enrolled and underwent successful sample collection for analysis. Three patients had incomplete datasets because of equipment malfunction and one elected not to participate. Seven patients had open tibia fractures, and one patient had an open fibula fracture associated with a closed tibia fracture. There were six Gustilo-Anderson Type II injuries and two Type IIIA injuries. Empiric antibiotics were administered in the prehospital setting or in the emergency department according to institutional protocol. When patients were taken to the operating room, a 2-g intravenous dose of cefazolin was administered. After surgical debridement, fracture stabilization, and wound closure, a microdialysis catheter was placed transdermally into the injury zone (within 5 cm of the fracture site) and a second catheter was placed in the contralateral uninjured (control) limb. Additional doses of cefazolin were administered every 8 hours postoperatively. Baseline and periodic interstitial fluid and whole blood (plasma) samples were collected in the operating room and at prespecified times for 24 hours postoperatively. Free cefazolin in the interstitial fluid and plasma samples were analyzed by ultra-high-performance liquid chromatography using C 18 column separation with quadrupole time-of-flight mass spectrometry detection. Data from the second postoperative dose of cefazolin were used to characterize pharmacokinetic parameters through a noncompartmental analysis using time-concentration curves of free cefazolin and assuming first-order elimination. For pharmacodynamic analyses, the modal cefazolin minimum inhibitory concentration (MIC) of Staphylococcus aureus (1 µg/mL) was used. RESULTS: With the samples available, no difference was observed in the median free cefazolin exposure over 24 hours ( f area under the curve [AUC] 0→24hrs ) between injured limbs (352 µg∙hr/mL [IQR 284 to 594 µg∙hr/mL]) and uninjured limbs (341 µg∙hr/mL [IQR 263 to 438 µg∙hr/mL]; p = 0.64). The median time to achieve the maximum concentration of free cefazolin ( f T max ) for injured limbs was delayed (2.7 hours [IQR 2.2 to 3.1 hours]) compared with control limbs (1.7 hours [IQR 1.2 to 2.0 hours]; p = 0.046). The time to the maximum concentration for plasma was not different from that of control limbs (p = 0.08). The time the cefazolin concentration was above the modal S. aureus MIC (T > MIC) in the injured and control limbs over 24 hours was 100% (IQR 100% to 100%) and 100% (IQR 97% to 100%), respectively. CONCLUSION: These preliminary findings suggest that current prophylactic cefazolin dosing regimens result in successful antibiotic delivery to the traumatized limb in moderately severe open fractures. Although cefazolin delivery to open-fracture wound beds was delayed compared with healthy tissues, the cefazolin concentration was sustained above the European Union Committee Antimicrobial Susceptibility Testing modal MIC for S. aureus , demonstrating a high likelihood of a prophylactic antimicrobial environment at an open fracture site with this empiric antimicrobial regimen. Importantly, patients in this analysis had Gustilo-Anderson Types II and IIIA injuries. Further research with a larger patient cohort is needed to determine whether antibiotic delivery to traumatized soft tissues in patients with higher-grade open fractures (Gustilo-Anderson Types IIIB and IIIC) demonstrates similar pharmacokinetic characteristics. LEVEL OF EVIDENCE: Level II, therapeutic study.

Meropenem pharmacokinetics in critically ill patients with or without burn treated with or without continuous veno-venous haemofiltration.

INTRODUCTION: Severe burn injury involves widespread skin and tissue damage leading to systemic inflammation, hypermetabolism and multi-organ failure. The hypermetabolic phase of burn injury has been associated with increased systemic antibiotic clearance; however, critical illness in the absence of burn may also induce similar physiologic changes. Continuous renal replacement therapy (CRRT) is often implemented in critically ill patients and may also affect antibiotic clearance. Although the pharmacokinetics (PK) of meropenem has been described in both the burn and non-burn critically ill populations, direct comparative data is lacking. METHODS: For this study, we evaluated PK parameters of meropenem from 23 critically ill patients, burn or non-burn, treated with or without continuous veno-venous haemofiltration (CVVH) to determine the contribution of burn and CVVH to the variability of therapeutic meropenem levels. RESULTS: A two-compartment model best described the data and revealed creatinine clearance (CrCl) and total burn surface area (TBSA) as significant covariates on clearance (CL) and peripheral volume of distribution (Vp), respectively. Of interest, non-burn patients on CVVH displayed an overall lower inherent CL as compared to burn patients on CVVH (6.43 vs. 12.85 L/h). Probability of target attainment (PTA) simulations revealed augmented renal clearance (ARC) may necessitate dose adjustments, but TBSA and CVVH would not. CONCLUSIONS: We recommend a standard dose of 1000 mg every 8 hours; however, if ARC is suspected, or the severity of illness requires a more stringent therapeutic target, we recommend a loading dose of 1000-2000 mg infused over 30 minutes to 1 hour followed by continuous infusion (3000-6000 mg over 24 hours), or intermittent infusion of 2000 mg every 8 hours.

Pharmacokinetics of piperacillin and tazobactam in critically Ill patients treated with continuous kidney replacement therapy: A mini-review and population pharmacokinetic analysis.

WHAT IS KNOWN AND OBJECTIVE: Timely and appropriate dosing of antibiotics is essential for the treatment of bacterial sepsis. Critically ill patients treated with continuous kidney replacement therapy (CKRT) often have physiologic derangements that affect pharmacokinetics (PK) of antibiotics and dosing may be challenging. We sought to aggregate previously published piperacillin and tazobactam (pip-tazo) pharmacokinetic data in critically ill patients undergoing CKRT to better understand pharmacokinetics of pip-tazo in this population and better inform dosing. METHODS: The National Library of Medicine Database was searched for original research containing piperacillin or tazobactam clearance (CL) or volume of distribution (V) estimates in patients treated with CKRT. The search yielded 77 articles, of which 26 reported suitable estimates of CL or V. Of the 26 articles, 10 for piperacillin and 8 for tazobactam had complete information suitable for population pharmacokinetic modelling. Also included in the analysis was piperacillin and tazobactam PK data from 4 critically ill patients treated with CKRT in the Military Health System, 2 with burn and 2 without burn. RESULTS AND DISCUSSION: Median and range of literature reported PK parameters for piperacillin (CL 2.76 L/hr, 1.4-7.92 L/hr, V 31.2 L, 16.77-42.27 L) and tazobactam (CL 2.34 L/hr, 0.72-5.2 L/hr, V 36.6 L, 26.2-58.87 L) were highly consistent with population estimates (piperacillin CL 2.7 L/hr, 95%CI 1.99-3.41 L/hr, V 25.83 22.07-29.59 L, tazobactam CL 2.49 L/hr, 95%CI 1.55-3.44, V 30.62 95%CI 23.7-37.54). The proportion of patients meeting pre-defined pharmacodynamic (PD) targets (median 88.7, range 71%-100%) was high despite significant mortality (median 44%, range 35%-60%). High mortality was predicted by baseline severity of illness (median APACHE II score 23, range 21-33.25). Choice of lenient or strict PD targets (ie 100%fT >MIC or 100%fT >4XMIC) had the largest impact on probability of target attainment (PTA), whereas presence or intensity of CKRT had minimal impact on PTA. WHAT IS NEW AND CONCLUSION: Pip-tazo overexposure may be associated with increased mortality, although this is confounded by baseline severity of illness. Achieving adequate pip-tazo exposure is essential; however, risk of harm from overexposure should be considered when choosing a PD target and dose. If lenient PD targets are desired, doses of 2250-3375 mg every 6 h are reasonable for most patients receiving CKRT. However, if a strict PD target is desired, continuous infusion (at least 9000-13500 mg per day) may be required. However, some critically ill CKRT populations may need higher or lower doses and dosing strategies should be tailored to individuals based on all available clinical data including the specific critical care setting.

Analysis of Nonbattle Deaths Among U.S. Service Members in the Deployed Environment.

OBJECTIVE: Describe etiologies and trends in non-battle deaths (NBD) among deployed U.S. service members to identify areas for prevention. BACKGROUND: Injuries in combat are categorized as battle (result of hostile action) or nonbattle related. Previous work found that one-third of injured US military personnel in Iraq and Afghanistan had nonbattle injuries and emphasized prevention. NBD have not yet been characterized. METHODS: U.S. military casualty data for Iraq and Afghanistan from 2001 to 2014 were obtained from the Defense Casualty Analysis System (DCAS) and the Department of Defense Trauma Registry (DoDTR). Two databases were used because DoDTR does not capture prehospital deaths, while DCAS does not contain clinical details. Nonbattle injuries and NBD were identified, etiologies classified, and NBD trends were assessed using a weighted moving average and time-series analysis with autoregressive integrated moving average. Future NBD rates were forecast. RESULTS: DCAS recorded 59,799 casualties; 21.0% (n = 1431) of all deaths (n = 6745) were NBD. DoDTR recorded 29,958 casualties; 11.5% (n = 206) of all deaths (n = 1788) were NBD. After early fluctuations, NBD rates for both Iraq and Afghanistan stabilized at approximately 21%. Leading causes of NBD were gunshot wounds and vehicle accidents, accounting for 66%. Approximately 25% was self-inflicted. A 24% NBD rate was forecasted from 2015 through 2025. CONCLUSIONS: Approximately 1 in 5 deaths were NBD. The majority were potentially preventable, including a significant proportion of self-inflicted injuries. A single comprehensive data repository would facilitate future mortality monitoring and performance improvement. These data may assist military leaders with implementing targeted safety strategies.

Evidence for misleading decision support in characterizing differences in tolerance to reduced central blood volume using measurements of tissue oxygenation.

BACKGROUND: The physiological response to hemorrhage includes vasoconstriction in an effort to shunt blood to the heart and brain. Hemorrhaging patients can be classified as "good" compensators who demonstrate high tolerance (HT) or "poor" compensators who manifest low tolerance (LT) to central hypovolemia. Compensatory vasoconstriction is manifested by lower tissue oxygen saturation (StO2 ), which has propelled this measure as a possible early marker of shock. The compensatory reserve measurement (CRM) has also shown promise as an early indicator of decompensation. METHODS: Fifty-one healthy volunteers (37% LT) were subjected to progressive lower body negative pressure (LBNP) as a model of controlled hemorrhage designed to induce an onset of decompensation. During LBNP, CRM was determined by arterial waveform feature analysis. StO2 , muscle pH, and muscle H+ concentration were calculated from spectrum using near-infrared spectroscopy (NIRS) on the forearm. RESULTS: These values were statistically indistinguishable between HT and LT participants at baseline (p ≥ 0.25). HT participants exhibited lower (p = 0.01) StO2 at decompensation compared to LT participants. CONCLUSIONS: Lower StO2 measured in patients during low flow states associated with significant hemorrhage does not necessarily translate to a more compromised physiological state, but may reflect a greater resistance to the onset of shock. Only the CRM was able to distinguish between HT and LT participants early in the course of hemorrhage, supported by a significantly greater ROC AUC (0.90) compared with STO2 (0.68). These results support the notion that measures of StO2 could be misleading for triage and resuscitation decision support.

Differentiating compensatory mechanisms associated with low tolerance to central hypovolemia in women.

Women generally display lower tolerance to acute central hypovolemia than men. The measurement of compensatory reserve (CRM) is a novel metric that provides information about the sum total of all mechanisms that together work to compensate for the relative blood volume deficit. Hemodynamic decompensation occurs with depletion of the CRM (i.e., 0% CRM). In the present study, we hypothesized that the lower tolerance to progressive central hypovolemia reported in women can be explained by a faster reduction rate in CRM compared with men rather than sex differences in absolute integrated compensatory responses. Continuous, noninvasive measures of CRM were collected from 208 healthy volunteers (107 men and 85 women) who underwent progressive stepwise central hypovolemia induced by lower body negative pressure to the point of presyncope. Comparisons revealed shorter ( P < 0.01) times in female participants compared with male participants to reach 30% and 0% CRM. Similarly, the lower body negative pressure level, represented by the cumulative stress index, was less at 30% and 0% CRM in women compared with men ( P < 0.01). Changes in hemodynamic responses and frequency-domain data (oscillations in cerebral blood flow velocity and mean arterial blood pressure) were similar between men and women at 0% CRM ( P > 0.05). We conclude that compensatory responses to central hypovolemia in women were similar to men but were depleted at a faster rate compared with men. The earlier depletion of the compensatory reserve in women appears to be influenced by failure to maintain adequate cerebral oxygen delivery. NEW & NOTEWORTHY We compared hemodynamic and metabolic responses in men and women to experimentally controlled reductions in central blood volume at physiologically equivalent levels of compensatory reserve. We corroborated previous findings that females have lower tolerance to central hypovolemia than males but demonstrated for the first time that compensatory responses are similar. Our findings suggest lower tolerance to central hypovolemia in women results from reaching critical cerebral delivery of oxygen faster than men.

Molecular Detection of Filamentous Fungi in Formalin-Fixed Paraffin-Embedded Specimens in Invasive Fungal Wound Infections Is Feasible with High Specificity.

Trauma-related invasive fungal wound infections (IFIs) are associated with significant morbidity and mortality. Early identification and treatment are critical. Traditional identification methods (e.g., fungal cultures and histopathology) can be delayed and insensitive. We assessed a PCR-based sequencing assay for rapid identification of filamentous fungi in formalin-fixed paraffin-embedded (FFPE) specimens obtained from combat casualties injured in Afghanistan. Blinded FFPE specimens from cases (specimens positive on histopathology) and controls (specimens negative on histopathology) were submitted for evaluation with a panfungal PCR. The internal transcribed spacer 2 (ITS2) region of the fungal ribosomal repeat was amplified and sequenced. The PCR results were compared with findings from histopathology and/or culture. If injury sites contributed multiple specimens, findings for the site were collapsed to the site level. We included 64 case subjects (contributing 95 sites) and 102 controls (contributing 118 sites). Compared to histopathology, panfungal PCR was specific (99%), but not as sensitive (63%); however, sensitivity improved to 83% in specimens from sites with angioinvasion. Panfungal PCR identified fungi of the order Mucorales in 33 of 44 sites with angioinvasion (75%), whereas fungal culture was positive in 20 of 44 sites (45%). Saksenaea spp. were the dominant fungi identified by PCR in specimens from angioinvasion sites (57%). Panfungal PCR is specific, albeit with lower sensitivity, and performs better at identifying fungi of the order Mucorales than culture. DNA sequencing offers significant promise for the rapid identification of fungal infection in trauma-related injuries, leading to more timely and accurate diagnoses.

In Vitro activity of Manuka Honey and polyhexamethylene biguanide on filamentous fungi and toxicity to human cell lines.

Soft-tissue invasive fungal infections are increasingly recognized as significant entities directly contributing to morbidity and mortality. They complicate clinical care, requiring aggressive surgical debridement and systemic antifungal therapy. To evaluate new topical approaches to therapy, we examined the antifungal activity and cytotoxicity of Manuka Honey (MH) and polyhexamethylene biguanide (PHMB). The activities of multiple concentrations of MH (40%, 60%, 80%) and PHMB (0.01%, 0.04%, 0.1%) against 13 clinical mould isolates were evaluated using a time-kill assay between 5 min and 24 h. Concentrations were selected to represent current clinical use. Cell viability was examined in parallel for human epidermal keratinocytes, dermal fibroblasts and osteoblasts, allowing determination of the 50% viability (LD50) concentration. Antifungal activity of both agents correlated more closely with exposure time than concentration. Exophiala and Fusarium growth was completely suppressed at 5 min for all PHMB concentrations, and at 12 and 6 h, respectively, for all MH concentrations. Only Lichtheimia had persistent growth to both agents at 24 h. Viability assays displayed concentration-and time-dependent toxicity for PHMB. For MH, exposure time predicted cytotoxicity only when all cell types were analyzed in aggregate. This study demonstrates that MH and PHMB possess primarily time-dependent antifungal activity, but also exert in vitro toxicity on human cells which may limit clinical use. Further research is needed to determine ideal treatment strategies to optimize antifungal activity against moulds while limiting cytotoxicity against host tissues in vivo.

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections.

Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both tissues and individual cell lines. Collectively our findings demonstrate that, similar to other polyamines, norspermidine displays both inhibitory and dispersive activities on biofilms of clinical multidrug-resistant bacterial isolates, in particular for strains of A. baumannii. Additionally our findings suggest that direct application may be considered on tissues, albeit for limited exposure times.

The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members.

BACKGROUND: The role of microbial colonization in disease is complex. Novel molecular tools to detect colonization offer theoretical improvements over traditional methods. We evaluated PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) as a screening tool to study colonization of healthy military service members. METHODS: We assessed 101 healthy Soldiers using PCR/ESI-TOF-MS on nares, oropharynx, and groin specimens for the presence of gram-positive and gram-negative bacteria (GNB), fungi, and antibiotic resistance genes. A second set of swabs was processed by traditional culture, followed by identification using the BD Phoenix automated system; comparison between PCR/ESI-TOF-MS and culture was carried out only for GNB. RESULTS: Using PCR/ESI-TOF-MS, at least one colonizing organism was found on each individual: mean (SD) number of organisms per subject of 11.8(2.8). The mean number of organisms in the nares, groin and oropharynx was 3.8(1.3), 3.8(1.4) and 4.2(2), respectively. The most commonly detected organisms were aerobic gram-positive bacteria: primarily coagulase-negative Staphylococcus (101 subjects: 341 organisms), Streptococcus pneumoniae (54 subjects: 57 organisms), Staphylococcus aureus (58 subjects: 80 organisms) and Nocardia asteroides (45 subjects: 50 organisms). The mecA gene was found in 96 subjects. The most commonly found GNB was Haemophilus influenzae (20 subjects: 21 organisms) and the most common anaerobe was Propionibacterium acnes (59 subjects). Saccharomyces species (30 subjects) were the most common fungi detected. Only one GNB (nares E. coli) was identified in the same subject by both diagnostic systems. CONCLUSION: PCR/ESI-TOF-MS detected common colonizing organisms and identified more typically-virulent bacteria in asymptomatic, healthy adults. PCR/ESI-TOF-MS appears to be a useful method for detecting bacterial and fungal organisms, but further clinical correlation and validation studies are needed.

Preliminary method for direct quantification of colistin methanesulfonate by attenuated total reflectance Fourier transform infrared spectroscopy.

Colistin use has increased in response to the advent of infections caused by multidrug-resistant organisms. It is administered parenterally as an inactive prodrug, colistin methanesulfonate (CMS). Various formulations of CMS and labeling conventions can lead to confusion about colistin dosing, and questions remain about the pharmacokinetics of CMS. Since CMS does not have strong UV absorbance, current methods employ a laborious process of chemical conversion to colistin followed by precolumn derivatization to detect formed colistin by high-performance liquid chromatography. Here, we report a method for direct quantification of colistin methanesulfonate by attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR).

In vitro study of the variable effects of proton pump inhibitors on voriconazole.

Voriconazole is a broad-spectrum antifungal agent used for the treatment of severe fungal infections. Maintaining therapeutic concentrations of 1 to 5.5 µg/ml is currently recommended to maximize the exposure-response relationship of voriconazole. However, this is challenging, given the highly variable pharmacokinetics of the drug, which includes metabolism by cytochrome P450 (CYP450) isotypes CYP2C19, CYP3A4, and CYP2C9, through which common metabolic pathways for many medications take place and which are also expressed in different isoforms with various metabolic efficacies. Proton pump inhibitors (PPIs) are also metabolized through these enzymes, making them competitive inhibitors of voriconazole metabolism, and coadministration with voriconazole has been reported to increase total voriconazole exposure. We examined the effects of five PPIs (rabeprazole, pantoprazole, lansoprazole, omeprazole, and esomeprazole) on voriconazole concentrations using four sets of human liver microsomes (HLMs) of different CYP450 phenotypes. Overall, the use of voriconazole in combination with any PPI led to a significantly higher voriconazole yield compared to that achieved with voriconazole alone in both pooled HLMs (77% versus 59%; P < 0.001) and individual HLMs (86% versus 76%; P < 0.001). The mean percent change in the voriconazole yield from that at the baseline after PPI exposure in pooled microsomes ranged from 22% with pantoprazole to 51% with esomeprazole. Future studies are warranted to confirm whether and how the deliberate coadministration of voriconazole and PPIs can be used to boost voriconazole levels in patients with difficult-to-treat fungal infections.

Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism.

Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 µg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity.

Colistin pharmacokinetics in burn patients during continuous venovenous hemofiltration.

While colistin is considered a last resort for the treatment of multidrug-resistant Gram-negative bacterial infections, there has been an increase in its use due to the increasing prevalence of drug-resistant infections worldwide. The pharmacology of colistin is complex, and pharmacokinetic data are limited, especially in patients requiring renal replacement therapy. As a result, dosing for patients who require renal replacement remains a challenge. Here, we present pharmacokinetic data for colistin from two burn patients (37 and 68 years old) infected with colistin-susceptible isoclonal Acinetobacter baumannii and receiving continuous venovenous hemofiltration (CVVH). To our knowledge, we are the first to examine data from before and during CVVH (for one patient), allowing analysis of the effect of CVVH on colistin pharmacokinetics. Pharmacokinetic/pharmacodynamic analysis indicated that a dose increase from 1.5 to 2.2 mg/kg of body weight colistin base activity on CVVH was insufficient to satisfy the target parameter of an AUC24/MIC (area under the concentration-time curve over 24 h in the steady state divided by the MIC) of ≥ 60 at an MIC of ≥ 1 µg/ml in one patient with residual endogenous renal function. Plasma concentrations of colistin ranged from 0 to 15 µg/ml, with free colistin levels ranging from 0.4 to 2.2 µg/ml. While both patients resolved their clinical infections and survived to discharge, colistin-resistant colonizing isolates resulted from therapy in one patient. The variabilities observed in colistin concentrations and pharmacokinetic characteristics highlight the importance of pharmacokinetic monitoring of antibiotics in patients undergoing renal replacement therapy.

In vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells.

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

Antifungal wound penetration of amphotericin and voriconazole in combat-related injuries: case report.

BACKGROUND: Survivors of combat trauma can have long and challenging recoveries, which may be complicated by infection. Invasive fungal infections are a rare but serious complication with limited treatment options. Currently, aggressive surgical debridement is the standard of care, with antifungal agents used adjunctively with uncertain efficacy. Anecdotal evidence suggests that antifungal agents may be ineffective in the absence of surgical debridement, and studies have yet to correlate antifungal concentrations in plasma and wounds. CASE PRESENTATION: Here we report the systemic pharmacokinetics and wound effluent antifungal concentrations of five wounds from two male patients, aged 28 and 30 years old who sustained combat-related blast injuries in southern Afghanistan, with proven or possible invasive fungal infection. Our data demonstrate that while voriconazole sufficiently penetrated the wound resulting in detectable effluent levels, free amphotericin B (unbound to plasma) was not present in wound effluent despite sufficient concentrations in circulating plasma. In addition, considerable between-patient and within-patient variability was observed in antifungal pharmacokinetic parameters. CONCLUSION: These data highlight the need for further studies evaluating wound penetration of commonly used antifungals and the role for therapeutic drug monitoring in providing optimal care for critically ill and injured war fighters.

Clinical infectious outcomes associated with biofilm-related bacterial infections: a retrospective chart review.

BACKGROUND: Biofilms are associated with persistent infection. Reports characterizing clinical infectious outcomes and patient risk factors for colonization or infection with biofilm forming isolates are scarce. Our institution recently published a study examining the biofilm forming ability of 205 randomly selected clinical isolates. This present study aims to identify potential risk factors associated with these isolates and assess clinical infectious outcomes. METHODS: 221 clinical isolates collected from 2005 to 2012 and previously characterized for biofilm formation were studied. Clinical information from the associated patients, including demographics, comorbidities, antibiotic usage, laboratory values, and clinical infectious outcomes, was determined retrospectively through chart review. Duplicate isolates and non-clinical isolates were excluded from analysis. Associations with biofilm forming isolates were determined by univariate analysis and multivariate analysis. RESULTS: 187 isolates in 144 patients were identified for analysis; 113 were biofilm producers and 74 were not biofilm producers. Patients were primarily male (78 %) military members (61 %) with combat trauma (52 %). On multivariate analysis, the presence of methicillin-resistant Staphylococcus aureus (p < 0.01, OR 5.09, 95 % CI 1.12, 23.1) and Pseudomonas aeruginosa (p = 0.02, OR 3.73, 95 % CI 1.46, 9.53) were the only characteristics more likely to be present in the biofilm producing isolate group. Infectious outcomes of patients with non-biofilm forming isolates, including cure, relapse/reinfection, and chronic infection, were similar to infectious outcomes of patients with biofilm-forming isolates. Mortality with initial infection was higher in the biofilm producing isolate group (16 % vs 5 %, p = 0.01) but attributable mortality was low (1 of 14). No characteristics examined in this study were found to be associated with relapse/reinfection or chronic infection on multivariate analysis. CONCLUSIONS: Bacteria species, but not clinical characteristics, were associated with biofilm formation on multivariate analysis. Biofilm forming isolates and non-biofilm forming isolates had similar infectious outcomes in this study.

Biofilm formation by clinical isolates and its relevance to clinical infections.

Reports of biofilms have increased exponentially in the scientific literature over the past two decades, yet the vast majority of these are basic science investigations with limited clinical relevance. Biofilm studies involving clinical isolates are most often surveys of isolate collections, but suffer from lack of standardization in methodologies for producing and assessing biofilms. In contrast, more informative clinical studies correlating biofilm formation to patient data have infrequently been reported. In this chapter, biofilm surveys of clinical isolates of aerobic and anaerobic bacteria, mycobacteria, and Candida are reviewed, as well as those pertaining to the unique situation of cystic fibrosis. In addition, the influence of host components on in vitro biofilm formation, as well as published studies documenting the clinical impact of biofilms in human infections, are presented.

D-amino acids enhance the activity of antimicrobials against biofilms of clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa.

Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of D-amino acids (D-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of D-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. D-Met, D-Phe, and D-Trp at concentrations of ≥ 5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (D-Met/D-Phe/D-Trp). When combined with D-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 µg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of D-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 µg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of D-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.

Dakin solution alters macrophage viability and function.

BACKGROUND: Macrophages are important in wound defense and healing. Dakin's solution (DS), buffered sodium hypochlorite, has been used since World War I as a topical antimicrobial for wound care. DS has been shown to be toxic to host cells, but effects on immune cells are not well documented. MATERIALS AND METHODS: DS at 0.5%, 0.125%, and ten-fold serial dilutions from 0.25%-0.00025% were evaluated for cellular toxicity on murine macrophages (J774A.1). The effect of DS on macrophage adhesion, phagocytosis, and generation of reactive oxygen species was examined. Macrophage polarization following DS exposure was determined by gene expression using quantitative real-time polymerase chain reaction. RESULTS: Concentrations of DS >0.0025% reduced macrophage viability to <5% in exposure times as short as 30 s. Similarly, phagocytosis of Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus flavus were significantly reduced at all tested concentrations by macrophages pretreated with DS. H2O2 production was reduced by 8%-38% following treatment with 0.00025%-0.125% DS. Macrophage adherence was significantly increased with >0.0025% DS after 15 min of exposure compared with controls. Quantitative real-time polymerase chain reaction demonstrated that DS exposure resulted in classical macrophage activation, with increased expression of inducible nitric oxide synthase 2, interferon-γ, and interleukin (IL)-1ß. CONCLUSIONS: DS at clinically used concentrations (0.025%-0.25%) was detrimental to macrophage survival and function. For optimal clinical use, understanding the impact of DS on macrophages is important as depletion may result in impaired pathogen clearance and delayed healing. These findings indicate that 0.00025% DS is a safe starting dose; however, optimal use of DS requires further validation with in vivo models.