Single-step extraction of fluconazole from plasma by ultra-filtration for the measurement of its free concentration by high performance liquid chromatography.

High performance liquid chromatography (HPLC) is the reference method for measuring concentrations of antimicrobials in blood. This technique requires careful sample preparation. Protocols using organic solvents and/or solid extraction phases are time consuming and entail several manipulations, which can lead to partial loss of the determined compound and increased analytical variability. Moreover, to obtain sufficient material for analysis, at least 1 ml of plasma is required. This constraint makes it difficult to determine drug levels when blood sample volumes are limited. However, drugs with low plasma-protein binding can be reliably extracted from plasma by ultra-filtration with a minimal loss due to the protein-bound fraction. This study validated a single-step ultra-filtration method for extracting fluconazole (FLC), a first-line antifungal agent with a weak plasma-protein binding, from plasma to determine its concentration by HPLC. Spiked FLC standards and unknowns were prepared in human and rat plasma. Samples (240 microl) were transferred into disposable microtube filtration units containing cellulose or polysulfone filters with a 5 kDa cut-off. After centrifugation for 60 min at 15000g, FLC concentrations were measured by direct injection of the filtrate into the HPLC. Using cellulose filters, low molecular weight proteins were eluted early in the chromatogram and well separated from FLC that eluted at 8.40 min as a sharp single peak. In contrast, with polysulfone filters several additional peaks interfering with the FLC peak were observed. Moreover, the FLC recovery using cellulose filters compared to polysulfone filters was higher and had a better reproducibility. Cellulose filters were therefore used for the subsequent validation procedure. The quantification limit was 0.195 mgl(-1). Standard curves with a quadratic regression coefficient > or = 0.9999 were obtained in the concentration range of 0.195-100 mgl(-1). The inter and intra-run accuracies and precisions over the clinically relevant concentration range, 1.875-60 mgl(-1), fell well within the +/-15% variation recommended by the current guidelines for the validation of analytical methods. Furthermore, no analytical interference was observed with commonly used antibiotics, antifungals, antivirals and immunosuppressive agents. Ultra-filtration of plasma with cellulose filters permits the extraction of FLC from small volumes (240 microl). The determination of FLC concentrations by HPLC after this single-step procedure is selective, precise and accurate.

Process and Outcome Evaluation of the STAR (Sisters Together Achieving Recovery) Program, September, 2010

In July of 2009, the Division of Criminal and Juvenile Justice Planning (CJJP) received Byrne Justice Assistance Grant/American Recovery and Reinvestment Act funding from the Governor’s Office of Drug Control Policy to conduct a process and outcome evaluation of the STAR (Sisters Together Achieving Recovery) program housed at the Iowa Correctional Institution for Women (ICIW) in Mitchellville, Iowa. The STAR Program is a licensed inpatient substance abuse treatment program that utilizes a Therapeutic Community model (TC). All offenders exiting the STAR program between October 1, 2004 and June 30, 2008 were included in the study (n=173). A comparison sample was drawn of offenders exiting the ICIW during the same release time frame with identified but untreated substance abuse needs (n= 173). March 31, 2010 was designated as the cut-off date for the study. This yielded an average post-program follow-up time of 3.1 years. The STAR group was further divided into two groups by time of program exit. Participants exiting the program between October 1, 2004 and June 30, 2006 were designated as STAR 1 (n=78) and those exiting the program between July 1, 2006 and June 30, 2008 were designated as STAR 2 (n=95). In order to have comparable tracking time between STAR groups, tracking time for STAR 1 concluded July 31, 2008. This yielded an average post release follow-up time of 2.4 years for both groups. Demographic, Program, Intervention, and Outcome data were examined. Comparisons were made between groups as well as categories of participation.

Acute multivessel revascularization improves 1-year outcome in ST-elevation myocardial infarction: a nationwide study cohort from the AMIS Plus registry.

BACKGROUND: The optimal strategy for percutaneous coronary intervention (PCI) of ST-segment elevation myocardial infarction (STEMI) in multi-vessel disease (MVD), i.e., multi-vessel PCI (MV-PCI) vs. PCI of the infarct-related artery only (IRA-PCI), still remains unknown. METHODS: Patients of the AMIS Plus registry admitted with an acute coronary syndrome were contacted after a median of 378 days (interquartile range 371-409). The primary end-point was all-cause death. The secondary end-point included all major adverse cardiovascular and cerebrovascular events (MACCE) including death, re-infarction, re-hospitalization for cardiac causes, any cardiac re-intervention, and stroke. RESULTS: Between 2005 and 2012, 8330 STEMI patients were identified, of whom 1909 (24%) had MVD. Of these, 442 (23%) received MV-PCI and 1467 (77%) IRA-PCI. While all-cause mortality was similar in both groups (2.7% both, p>0.99), MACCE was significantly lower after MV-PCI vs. IRA-PCI (15.6% vs. 20.0%, p=0.038), mainly driven by lower rates of cardiac re-hospitalization and cardiac re-intervention. Patients undergoing MV-PCI with drug-eluting stents had lower rates of all-cause mortality (2.1% vs. 7.4%, p=0.026) and MACCE (14.1% vs. 25.9%, p=0.042) compared with those receiving bare metal stents (BMS). In multivariate analysis, MV-PCI (odds ratio, OR 0.69, 95% CI 0.51-0.93, p=0.017) and comorbidities (Charlson index ≥ 2; OR 1.42, 95% CI 1.05-1.92, p=0.025) were independent predictors for 1-year MACCE. CONCLUSION: In an unselected nationwide real-world cohort, an approach using immediate complete revascularization may be beneficial in STEMI patients with MVD regarding MACCE, specifically when drug-eluting stents are used, but not regarding mortality. This has to be tested in a randomized controlled trial.

Recurrence of status epilepticus: Prognostic role and outcome predictors.

OBJECTIVE: Predictors of morbidity and mortality after status epilepticus (SE) have been studied extensively in hospital- and population-based cohorts. However, little attention has been directed toward SE recurrence after an incident episode. We investigated clinical and demographic characteristics of patients presenting SE recurrence and its specific prognostic role. METHODS: In this observational cohort study, we screened our prospective registry of consecutive adults with SE between April 2006 and February 2014. Demographic and clinical data were compared between incident and recurrent SE episodes; risk of SE recurrence was assessed through survival analysis, and the prognostic role of SE recurrence with multivariable logistic regressions. RESULTS: Of the incident cohort (509 patients), 68 (13%) experienced recurrent SE. The cumulative recurrence rate over 4 years was 32%. Recurrence risk was significantly reduced after an acute SE etiology (hazard ratio [HR] 0.5, 95% confidence interval [CI] 0.31-0.82; p = 0.005), and was borderline increased in women (HR 1.59, 95% CI 0.97-2.65; p = 0.06). Although recurrent SE episodes showed lower morbidity and mortality, prognosis was independently related to Status Epilepticus Severity Score (STESS) and potentially fatal etiology, but not to SE recurrence. SIGNIFICANCE: This study provides class III evidence that SE recurrence involves a significant proportion of patients, and that recurrence risk is independently associated with chronic etiology and to a lesser extent with female gender. However, contrary to underlying cause and SE severity, SE recurrence per se does not independently correlate with outcome. Early identification of patients at higher risk of SE recurrence may influence their management during follow-up.

First aid in burn injuries with counterintuitive warm water improves outcome

Objective: Cooling is considered a panacea in burn injury. However, burn injuries are characterized by an ischemic zone prone to progression, a phenomenon that can substantially increase morbidity. Cold-induced vasoconstriction potentially aggravates ischemia and promotes progression. Therefore we compared the effect of warm (37°C) and cold (17°C) water on burn progression. Methods: The comb burn model creates 4 rectangular burned surfaces separated by 3 unburned interspaces that become necrotic if untreated. After heating in boiling water the template was applied for 60 seconds on 24 Wistar rats randomized into 3 groups: no treatment (CON); treatment for 20 minutes with cold water (17°C: CW) or warm water (37°C: WW). Burn progression in surface (planimetry) and Departmenth (histology), as well as microcirculatory perfusion (laser Doppler flowmetry) were assessed after 1h, as well as 1, 4, and 7 days. Results: Both CW and WW delayed burn progression without reducing the final burn Departmenth (deep dermis). In contrast, only WW but not CW increased dermal perfusion (81 ± 2% (WW) vs. 62 ± 2% (CW) and 63 ± 1% (CON), p< 0·05) already 1 hour after burn induction. The difference observed after one hour led to a complete flow recovery during the observation period and translated into increased interspace survival, respectively less necrosis with WW(65 ± 4% vs. 81 + 4% (CW) and 91 ± 2% (CON), p< 0·05) after 7 days. Conclusions: Application of warm water significantly improved dermal perfusion, increased interspace survival, and delayed burn progression.However it didn't alter the ultimate burn Departmenth of the actually burned area. Therefore, warm water can create a therapeutic window for targeted nonsurgical treatment of burn progression.

MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status.

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p < 0.001) using a training-set of 63 glioblastomas from homogenously treated patients, for whom MGMT methylation was previously shown to be predictive for outcome based on classification by methylation-specific PCR. MGMT-STP27 was successfully validated in an independent cohort of chemo-radiotherapy-treated glioblastoma patients (n = 50; kappa = 0.88; outcome, log-rank p < 0.001). Lower prevalence of MGMT methylation among CpG island methylator phenotype (CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM-450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types.

Diagnosis of pulmonary embolism by multidetector CT alone or combined with venous ultrasonography of the leg: a randomised non-inferiority trial.

BACKGROUND: Multislice CT (MSCT) combined with D-dimer measurement can safely exclude pulmonary embolism in patients with a low or intermediate clinical probability of this disease. We compared this combination with a strategy in which both a negative venous ultrasonography of the leg and MSCT were needed to exclude pulmonary embolism. METHODS: We included 1819 consecutive outpatients with clinically suspected pulmonary embolism in a multicentre non-inferiority randomised controlled trial comparing two strategies: clinical probability assessment and either D-dimer measurement and MSCT (DD-CT strategy [n=903]) or D-dimer measurement, venous compression ultrasonography of the leg, and MSCT (DD-US-CT strategy [n=916]). Randomisation was by computer-generated blocks with stratification according to centre. Patients with a high clinical probability according to the revised Geneva score and a negative work-up for pulmonary embolism were further investigated in both groups. The primary outcome was the 3-month thromboembolic risk in patients who were left untreated on the basis of the exclusion of pulmonary embolism by diagnostic strategy. Clinicians assessing outcome were blinded to group assignment. Analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT00117169. FINDINGS: The prevalence of pulmonary embolism was 20.6% in both groups (189 cases in DD-US-CT group and 186 in DD-CT group). We analysed 855 patients in the DD-US-CT group and 838 in the DD-CT group per protocol. The 3-month thromboembolic risk was 0.3% (95% CI 0.1-1.1) in the DD-US-CT group and 0.3% (0.1-1.2) in the DD-CT group (difference 0.0% [-0.9 to 0.8]). In the DD-US-CT group, ultrasonography showed a deep-venous thrombosis in 53 (9% [7-12]) of 574 patients, and thus MSCT was not undertaken. INTERPRETATION: The strategy combining D-dimer and MSCT is as safe as the strategy using D-dimer followed by venous compression ultrasonography of the leg and MSCT for exclusion of pulmonary embolism. An ultrasound could be of use in patients with a contraindication to CT.

Outcome of subscapularis muscle release for shoulder contracture secondary to brachial plexus palsy at birth.

Children with unresolved brachial plexus palsy frequently develop a disabling internal rotation contracture of the shoulder. Several surgical options, including soft tissue procedures such as muscle releases and/or transfers, and bone operations such as humeral osteotomy are available to correct this deformity. This study describes the effect of subscapularis muscle release performed in isolation. Thirteen patients (5 boys, 8 girls) were reviewed at an average of 3.5 years after their surgery (range, 2-7 years). Their mean age at operation was 4.7 years (range, 1-8 years). Three children had C5-C6 palsies, 8 had C5-C7 palsies, and 2 had C5-C8 palsies. Postoperatively, patients presented significant gains in shoulder active lateral rotation (+49 degrees, from 5 to 54 degrees), active abduction (+30 degrees, from 63 to 93 degrees), active flexion (+46 degrees, from 98 to 144 degrees), and active extension (+23 degrees, from 7 to 30 degrees). Gains were also observed in passive range of motion, but of a lesser degree. Subscapularis muscle release is a procedure we found to have few significant complications and was highly effective in increasing active range of motion and restoring shoulder function.

Biomechanical concept and clinical outcome of dual mobility cups.

Dual mobility cup systems in total hip arthroplasty consist of a metal back with a non-constrained liner, in which a constrained standard head articulates. While superior stability of such implants in comparison with standard total hip replacements is assumed, it is the purpose of this study to outline the biomechanical concept of dual mobility cups and to describe implant survival and dislocation rate based on the series published in the English-speaking and Francophone literature. A growing body of evidence indicates reduced dislocation rates in primary and revision total hip arthroplasty and in selected tumour cases. The limited availability of studies evaluating long-term implant survival and existing concerns with regard to increased wear rates and aseptic loosening, leads to the conclusion that such implants have to be used with prudence, particularly in standard primary hip arthroplasty and in young patients.

Endocan measurement for the postmortem diagnosis of sepsis

The vascular endothelium has been shown to play a pivotal role in the pathophysiology of sepsis through the expression of surface proteins and secretion of soluble mediators. Endocan (endothelial cell-specific molecule-1), a 50-kDa dermatan sulfate proteoglycan, is expressed by endothelial cells in lung and kidney and can be detected at low levels in the serum of healthy subjects. Increased concentrations were described in patients with sepsis, severe sepsis and septic shock compared to healthy individuals, with serum concentrations related to the severity of illness. In the present study, we investigated endocan, procalcitonin and C-reactive protein in postmortem serum from femoral blood in a series of sepsis-related fatalities and control individuals who underwent medicolegal investigations. Endocan was also measured in pericardial fluid. Two study groups were prospectively formed, a sepsis-related fatalities group and a control group. The sepsis-related fatalities group consisted of sixteen forensic autopsy cases with documented clinical diagnosis of sepsis in vivo. The control group consisted of sixteen forensic autopsy cases with various noninfectious causes of death. Postmortem serum endocan concentrations were significantly higher in the sepsis group, with values ranging from 0.519ng/ml to 6.756ng/ml. In the control group, endocan levels were undetectable in eleven out of sixteen cases. The results of the data analysis revealed similar endocan concentrations in the pericardial fluid of both studied groups. Endocan can be considered a suitable biological parameter for the detection of sepsis-related deaths in forensic pathology routine.

Case complexity and clinical outcome in diabetes mellitus. A prospective study using the INTERMED.

The aim of this study was to assess a population of patients with diabetes mellitus by means of the INTERMED, a classification system for case complexity integrating biological, psychosocial and health care related aspects of disease. The main hypothesis was that the INTERMED would identify distinct clusters of patients with different degrees of case complexity and different clinical outcomes. Patients (n=61) referred to a tertiary reference care centre were evaluated with the INTERMED and followed 9 months for HbA1c values and 6 months for health care utilisation. Cluster analysis revealed two clusters: cluster 1 (62%) consisting of complex patients with high INTERMED scores and cluster 2 (38%) consisting of less complex patients with lower INTERMED. Cluster 1 patients showed significantly higher HbA1c values and a tendency for increased health care utilisation. Total INTERMED scores were significantly related to HbA1c and explained 21% of its variance. In conclusion, different clusters of patients with different degrees of case complexity were identified by the INTERMED, allowing the detection of highly complex patients at risk for poor diabetes control. The INTERMED therefore provides an objective basis for clinical and scientific progress in diabetes mellitus. Ongoing intervention studies will have to confirm these preliminary data and to evaluate if management strategies based on the INTERMED profiles will improve outcomes.

Clinical results of autologous infrainguinal revascularization using grafts originating distal to the femoral bifurcation in patients with mild inflow disease.

AIM: Chronic critical limb ischemia (CLI) often requires venous bypass grafting to distal arterial segments. However, graft patency is influenced by the length and quality of the graft and occasionally patients may have limited suitable veins. We investigated short distal bypass grafting from the superficial femoral or popliteal artery to the infrapopliteal, ankle or foot arteries, despite angiographic alterations of inflow vessels, providing that invasive pressure measurement at the site of the planned proximal anastomosis revealed an inflow-brachial pressure difference of <or=10 mmHg. METHODS: Four hundred and twenty-three consecutive infrainguinal bypass grafts were performed for CLI between June, 1999 and November, 2002 at our institution. All patients underwent preoperative clinical examination, arteriography and assessment of the veins by duplex ultrasound. The study group are patients in whom the proximal and distal anastomoses of the bypass are below the femoral bifurcation and the popliteal artery, respectively. Invasive arterial pressure measurements were recorded at the level of the planned proximal anastomosis which was performed at that level if the difference of the inflow-brachial pressure was <or=10 mmHg, irrespective of angiographic alterations of the inflow vessels proximal to the planned anastomosis. All patients had a clinical follow-up included a duplex examination of their graft, at 1 week, 3, 9 and 12 months and, thereafter, annually. No patient was lost to follow-up. RESULTS: Sixty-seven patients underwent 71 short distal bypass grafts in 71 limbs with reversed saphenous vein grafts in 52, in situ saphenous veins in 11, reversed cephalic vein in 1 and composite veins in 7, respectively. Surgical or endovascular interventions to improve inflow were required in 4 limbs (5.6%). The mean follow-up time was 22.5 months and the two-year survival was 92.5%. Primary and secondary patency rates at 2 years were 73% and 93%, respectively, and the limb salvage rate was 98.5%. CONCLUSION: In appropriately selected patients, short distal venous bypass grafts can be performed with satisfactory patency and limb salvage rates even in the presence of morphologic alterations of the inflow vessels providing that these are not hemodynamically significant, or can be corrected intraoperatively.

Measurement of multi-segment foot joint angles during gait using a wearable system.

Usually the measurement of multi-segment foot and ankle complex kinematics is done with stationary motion capture devices which are limited to use in a gait laboratory. This study aimed to propose and validate a wearable system to measure the foot and ankle complex joint angles during gait in daily conditions, and then to investigate its suitability for clinical evaluations. The foot and ankle complex consisted of four segments (shank, hindfoot, forefoot, and toes), with an inertial measurement unit (3D gyroscopes and 3D accelerometers) attached to each segment. The angles between the four segments were calculated in the sagittal, coronal, and transverse planes using a new algorithm combining strap-down integration and detection of low-acceleration instants. To validate the joint angles measured by the wearable system, three subjects walked on a treadmill for five minutes at three different speeds. A camera-based stationary system that used a cluster of markers on each segment was used as a reference. To test the suitability of the system for clinical evaluation, the joint angle ranges were compared between a group of 10 healthy subjects and a group of 12 patients with ankle osteoarthritis, during two 50-m walking trials where the wearable system was attached to each subject. On average, over all joints and walking speeds, the RMS differences and correlation coefficients between the angular curves obtained using the wearable system and the stationary system were 1 deg and 0.93, respectively. Moreover, this system was able to detect significant alteration of foot and ankle function between the group of patients with ankle osteoarthritis and the group of healthy subjects. In conclusion, this wearable system was accurate and suitable for clinical evaluation when used to measure the multi-segment foot and ankle complex kinematics during long-distance walks in daily life conditions.