Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Randomized controlled trial of urinE chemiStry guided aCute heArt faiLure treATmEnt (ESCALATE): Rationale and design.

Diuresis to achieve decongestion is a central aim of therapy in patients hospitalized for acute decompensated heart failure (ADHF). While multiple clinical trials have investigated initial diuretic strategies for a designated period of time, there is a paucity of evidence to guide diuretic titration strategies continued until decongestion is achieved. The use of urine chemistries (urine sodium and creatinine) in a natriuretic response prediction equation accurately estimates natriuresis in response to diuretic dosing, but a randomized clinical trial is needed to compare a urine chemistry-guided diuresis strategy with a strategy of usual care. The urinE chemiStry guided aCute heArt faiLure treATmEnt (ESCALATE) trial is designed to test the hypothesis that protocolized diuretic therapy guided by spot urine chemistry through completion of intravenous diuresis will be superior to usual care and improve outcomes over the 14 days following randomization. ESCALATE will randomize and obtain complete data on 450 patients with acute heart failure to a diuretic strategy guided by urine chemistry or a usual care strategy. Key inclusion criteria include an objective measure of hypervolemia with at least 10 pounds of estimated excess volume, and key exclusion criteria include significant valvular stenosis, hypotension, and a chronic need for dialysis. Our primary outcome is days of benefit over the 14 days after randomization. Days of benefit combines patient symptoms captured by global clinical status with clinical state quantifying the need for hospitalization and intravenous diuresis. CLINICAL TRIAL REGISTRATION: NCT04481919.

Current Use and Barriers to Point-of-Care Ultrasound in Primary Care: A National Survey of VA Medical Centers.

BACKGROUND: More primary care providers (PCPs) have begun to embrace the use of point-of-care ultrasound (POCUS), but little is known about how PCPs are currently using POCUS and what barriers exist. In this prospective study, the largest systematic survey of POCUS use among PCPs, we assessed the current use, barriers to use, program management, and training needs for POCUS in primary care. METHODS: We conducted a prospective observational study of all VA Medical Centers (VAMCs) between June 2019 and March 2020 using a web-based survey sent to all VAMC Chiefs of Staff and Chiefs of primary care clinics (PCCs). RESULTS: Chiefs of PCCs at 105 VAMCs completed the survey (82% response rate). Only 13% of PCCs currently use POCUS, and the most common applications used were bladder and musculoskeletal ultrasound. Desire for POCUS training exceeded current use, but lack of trained providers (78%), ultrasound equipment (66%), and funding for training (41%) were common barriers. Program infrastructure to support POCUS use was uncommon, and only 9% of VAMCs had local policies related to POCUS. Most PCC chiefs (64%) would support POCUS training. CONCLUSIONS: Current use of POCUS in primary care is low despite the recent growth of POCUS training in Internal Medicine residency programs. Investment in POCUS training and program infrastructure is needed to expand POCUS use in primary care and ensure adequate supervision of trainees.

Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

The c-Jun N-terminal kinase 3 (JNK3) is a stress-activated kinase primarily expressed in the brain and implicated as an early mediator of neuronal apoptosis. We sought to develop a PET tracer to visualize pathological JNK3 activation. Because regional JNK3 activation precedes apoptosis, such an imaging agent might enable the detection of "at risk" brain regions prior to neuronal death. We prepared a set of 19F-containing compounds on the basis of the reported aminopyrazoles. The candidate, F3, was tritiated and used in autoradiography experiments to demonstrate regional and temporal changes in JNK3 activation in a mouse model of Parkinson's disease. A significant increase in pJNK3 B max versus control animals in multiple brain regions was observed at 8 months, including the ventral midbrain. Pathological activation of JNK3 in these regions preceded statistically significant neuron loss. Analyses of brain concentrations of [18F]-F3 in naïve rats following intravenous injection revealed a small but detectable signal over the background, but was likely not sufficient to support PET imaging.

Current use, training, and barriers in point-of-care ultrasound in hospital medicine: A national survey of VA hospitals.

BACKGROUND: Point-of-care ultrasound (POCUS) can reduce procedural complications and improve the diagnostic accuracy of hospitalists. Currently, it is unknown how many practicing hospitalists use POCUS, which applications are used most often, and what barriers to POCUS use exist. OBJECTIVE: This study aimed to characterize current POCUS use, training needs, and barriers to use among hospital medicine groups (HMGs). DESIGN, SETTING, AND PARTICIPANTS: A prospective observational study of all Veterans Affairs (VA) medical centers was conducted between August 2019 and March 2020 using a web-based survey sent to all chiefs of HMGs. These data were compared to a similar survey conducted in 2015. RESULT: Chiefs from 117 HMGs were surveyed, with a 90% response rate. There was ongoing POCUS use in 64% of HMGs. From 2015 to 2020, procedural POCUS use decreased by 19%, but diagnostic POCUS use increased for cardiac (8%), pulmonary (7%), and abdominal (8%) applications. The most common barrier to POCUS use was lack of training (89%), and only 34% of HMGs had access to POCUS training. Access to ultrasound equipment was the least common barrier (57%). The proportion of HMGs with ≥1 ultrasound machine increased from 29% to 71% from 2015 to 2020. An average of 3.6 ultrasound devices per HMG was available, and 45% were handheld devices. CONCLUSION: From 2015 to 2020, diagnostic POCUS use increased, while procedural use decreased among hospitalists in the VA system. Lack of POCUS training is currently the most common barrier to POCUS use among hospitalists.

Development of a multisystem point of care ultrasound skills assessment checklist.

BACKGROUND: Many institutions are training clinicians in point-of-care ultrasound (POCUS), but few POCUS skills checklists have been developed and validated. We developed a consensus-based multispecialty POCUS skills checklist with anchoring references for basic cardiac, lung, abdominal, and vascular ultrasound, and peripheral intravenous line (PIV) insertion. METHODS: A POCUS expert panel of 14 physicians specializing in emergency, critical care, and internal/hospital medicine participated in a modified-Delphi approach to develop a basic POCUS skills checklist by group consensus. Three rounds of voting were conducted, and consensus was defined by ≥ 80% agreement. Items achieving < 80% consensus were discussed and considered for up to two additional rounds of voting. RESULTS: Thirteen POCUS experts (93%) completed all three rounds of voting. Cardiac, lung, abdominal, and vascular ultrasound checklists included probe location and control, basic machine setup, image quality and optimization, and identification of anatomical structures. PIV insertion included additional items for needle tip tracking. During the first round of voting, 136 (82%) items achieved consensus, and after revision and revoting, an additional 21 items achieved consensus. A total of 153 (92%) items were included in the final checklist. CONCLUSIONS: We have developed a consensus-based, multispecialty POCUS checklist to evaluate skills in image acquisition and anatomy identification for basic cardiac, lung, abdominal, and vascular ultrasound, and PIV insertion.

Systematic Sonography Looking for Occult Wounds: accuracy of an abdominal ultrasound adjunct in penetrating trauma.

BACKGROUND: Systematic Sonography Looking for Occult Wounds (SSLOW) in trauma is a novel technique for the evaluation of intra-abdominal wounds in penetrating trauma. No data exist regarding the effectiveness. The objective of this study was to evaluate the accuracy of the SSLOW exam. METHODS: This is a prospective collected case series conducted over a period of 10 months and took place at the Accident and Emergency Department (A&E) of the Georgetown Public Hospital Corporation (GPHC). The study enrolled patients presenting to the A&E who were 16 years old or greater with penetrating abdominal trauma. All patients with penetrating trauma received an E-FAST examination. If the E-FAST examination was negative, a SSLOW examination was completed. The sonographer evaluated for free fluid collection between the loops of bowel. The results of the SSLOW were compared to usual care (surgery consult, serial abdominal and E-FAST exams, laparotomy, and 7-day follow-up) and then categorized into four groups: true positive, false positive, true negative, and false negative. These results lead to four categorical values. From these results, sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratios were calculated. RESULTS: There were 5 (12%) true positives, 1 (2%) false positive, 37 (86%) true negatives, and zero (0%) false negative. The SSLOW was 100% sensitive (95% CI 5-100%) and 97% specificity (95% CI 74-96%). There was an 80% positive predictive value (95% CI 1.0-64% 95% CI) and 100% negative predictive value (95% CI 88-100%). The positive likelihood ratio was 8.4 (95% CI 3.69-19.1) and negative likelihood ratio was 0. CONCLUSION: The SSLOW examination may be a useful tool in the evaluation of penetrating abdominal injuries.

Observational descriptive study of ultrasound use and its impact on clinical decisions in the accident and emergency department at Georgetown public hospital corporation.

INTRODUCTION: Point-of-care ultrasound (POCUS) is physician-performed at the bedside, and it is a powerful diagnostic tool, especially in resource-limited emergency medicine healthcare settings. This study aims to quantify both the use of ultrasound and its impact on patient care at the Accident and Emergency Department at the Georgetown Public Hospital Corporation (GPHC). METHODS: This is a cross-sectional observational descriptive analysis of data collected for quality assessment in the GPHC Accident and Emergency Department. Over the course of two months, physicians were asked to record each ultrasound exam performed and record whether the ultrasound results changed patient disposition or the medication used in management. RESULTS: During the study period, there were 173 ultrasound data sheets collected representing 426 ultrasound studies. 196 studies were positive with pathologic findings (46.0%). The use of ultrasound in patient care either changed the patient's final disposition or medication 78.6% of the time. CONCLUSION: Ultrasound is used frequently at the Georgetown Public Hospital Corporation for a wide variety of applications. When utilized, POCUS frequently influenced patient care.

Fe-HBED Analogs: A Promising Class of Iron-Chelate Contrast Agents for Magnetic Resonance Imaging.

Contrast-enhanced magnetic resonance imaging is an essential tool for disease diagnosis and management; all marketed clinical magnetic resonance imaging (MRI) contrast agents (CAs) are gadolinium (Gd) chelates and most are extracellular fluid (ECF) agents. After intravenous injection, these agents rapidly distribute to the extracellular space and are also characterized by low serum protein binding and predominant renal clearance. Gd is an abiotic element with no biological recycling processes; low levels of Gd have been detected in the central nervous system and bone long after administration. These observations have prompted interest in the development of new MRI contrast agents based on biotic elements such as iron (Fe); Fe-HBED (HBED = N,N'-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid), a coordinatively saturated iron chelate, is an attractive MRI CA platform suitable for modification to adjust relaxivity and biodistribution. Compared to the parent Fe-HBED, the Fe-HBED analogs reported here have lower serum protein binding and higher relaxivity as well as lower relative liver enhancement in mice, comparable to that of a representative gadolinium-based contrast agent (GBCA). Fe-HBED analogs are therefore a promising class of non-Gd ECF MRI CA.

In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations.

Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre-clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150 mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration-time profiles (t0-240 min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R2 = 0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model.

Diagnostic use of lung ultrasound compared to chest radiograph for suspected pneumonia in a resource-limited setting.

BACKGROUND: Lung ultrasound is an effective tool for diagnosing pneumonia in developed countries. Diagnostic accuracy in resource-limited countries where pneumonia is the leading cause of death is unknown. The objective of this study was to evaluate the sensitivity of bedside lung ultrasound compared to chest X-ray for pneumonia in adults presenting for emergency care in a low-income country. METHODS: Patients presenting to the emergency department with suspected pneumonia were evaluated with bedside lung ultrasound, single posterioranterior chest radiograph, and computed tomography (CT). Using CT as the gold standard, the sensitivity of lung ultrasound was compared to chest X-ray for the diagnosis of pneumonia using McNemar's test for paired samples. Diagnostic characteristics for each test were calculated. RESULTS: Of 62 patients included in the study, 44 (71%) were diagnosed with pneumonia by CT. Lung ultrasound demonstrated a sensitivity of 91% compared to chest X-ray which had a sensitivity of 73% (p = 0.01). Specificity of lung ultrasound and chest X-ray were 61 and 50% respectively. CONCLUSIONS: Bedside lung ultrasound demonstrated better sensitivity than chest X-ray for the diagnosis of pneumonia in Nepal. TRIAL REGISTRATION: ClinicalTrials.gov, registration number NCT02949141 . Registered 31 October 2016.

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Estimating amounts of iron oxide from gradient echo images.

Rat legs directly injected with superparamagnetic iron oxide (SPIO) were studied by dual-echo, gradient-echo imaging. The amount of iron injected was estimated using a point dipole model for the SPIO injection site. Saturation magnetization of 6:1 PEG/amino modified silane-coated iron oxide particles with 5- to 6-nm core and 20-25 hydrodynamic diameter was approximately 110 emu/g of iron. Estimates of the amount of iron injected made from signal void volumes surrounding SPIO centers yielded erroneous results varying with sample orientation in the scanner and echo time (TE). For example, a 10 microL, 3-microg iron injection produced signal void volumes of 80 and 210 microL at TE of 9.8 and 25 ms, respectively, giving apparent iron contents of 6 +/- 1 and 10 +/- 2 microg respectively. A more effective approach uses the phase difference between two gradient recalled echo images. To estimate iron content, this approach fits the expected (3 cos(2)theta - 1)/(/r/3) spatial phase distribution to the observed phase differences. Extraneous phase effects made fitting phase at a single TE ineffective. With the dual echo method, 18 independent estimates were 2.48 +/- 0.26 microg std, independently of sample orientation. Estimates in empty control regions were -90 and -140 ng. A 1-microg injection indicated 0.5, 1.2, and 1.2 microg.

Mechanistic studies on DNA damage by minor groove binding copper-phenanthroline conjugates.

Copper-phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper-phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11-54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from beta-elimination of 2-deoxyribonolactone by copper-phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.

Alkane oxidation by osmium tetroxide.

Aqueous alkaline OsO4 at 85 degrees C oxidizes saturated alkanes, including primary, secondary, and tertiary C-H bonds. Isobutane affords tert-butanol in quantitative yield based on consumed OsO4. Cyclohexane is oxidized to a mixture of adipate and succinate. Ethane and propane are oxidized to acetate, which itself is further oxidized under the reaction conditions. A few turnovers of isobutane oxidation have been accomplished using NaIO4 as the terminal oxidant. The alkane oxidation is an example of ligand accelerated catalysis, as hydroxide binding to OsO4 is required for reaction. A concerted mechanism involving [3+2] addition of a C-H bond to two oxo groups of OsO4(OH)- is suggested, analogous to the pathways for dihydroxylation of alkenes by OsO4(L) and for addition of H2 to OsO4(L).

Repair of oxidized abasic sites by exonuclease III, endonuclease IV, and endonuclease III.

2-Deoxyribonolactone (L) and the C4'-oxidized abasic site (C4-AP) are produced by a variety of DNA-damaging agents. If not repaired, these lesions can be mutagenic. Exonuclease III and endonuclease IV are the major enzymes in E. coli responsible for 5'-incision of abasic sites (APs), the first steps in AP repair. Endonuclease III efficiently excises AP lesions via intermediate Schiff-base formation. Incision of L and C4-AP lesions by exonuclease III and endonuclease IV was determined under steady-state conditions using oligonucleotide duplexes containing the lesions at defined sites. An abasic lesion (AP) in an otherwise identical DNA sequence was incised by exonuclease III or endonuclease IV approximately 6-fold more efficiently than either of the oxidized abasic sites (L, C4-AP). Endonuclease IV incision efficiency of 2-deoxyribonolactone or C4-AP was independent of whether the lesion was opposite dA or dG. 2-Deoxyribonolactone is known to cross-link to endonuclease III (Hashimoto, M. (2001) J. Am. Chem. Soc. 123, 3161.). However, the C4-AP lesion is efficiently excised by endonuclease III. Oxidized abasic site repair by endonuclease IV and endonuclease III (C4-AP only) is approximately 100-fold less efficient than repair by exonuclease III. These results suggest that the first step of C4-AP and L oxidized abasic site repair will be the same as that of regular AP lesions in E. coli.

A minor groove binding copper-phenanthroline conjugate produces direct strand breaks via beta-elimination of 2-deoxyribonolactone.

Copper-phenanthroline complexes and their conjugates are useful reagents for studying nucleic acid interactions. Although DNA cleavage by such complexes was discovered more than 20 years ago, significant questions remain unanswered regarding the chemical mechanism(s) by which DNA is damaged. Kinetic evidence is provided, which demonstrates that the major pathway for DNA damage by a minor groove binding molecule conjugated to copper phenanthroline (6) involves C1'-oxidation. Additional experiments using 6 and a DNA substrate containing 2-deoxyribonolactone (1) show that direct strand breaks are produced via beta-elimination from 1. These studies support the original mechanism for DNA damage by copper phenanthroline put forth by Sigman and a more recent proposal concerning the mechanism for direct strand break formation.

Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone.

Oxidized abasic residues in DNA constitute a major class of radiation and oxidative damage. Free radical attack on the nucleotidyl C-1' carbon yields 2-deoxyribonolactone (dL) as a significant lesion. Although dL residues are efficiently incised by the main human abasic endonuclease enzyme Ape1, we show here that subsequent excision by human DNA polymerase beta is impaired at dL compared with unmodified abasic sites. This inhibition is accompanied by accumulation of a protein-DNA cross-link not observed in reactions of polymerase beta with unmodified abasic sites, although a similar form can be trapped by reduction with sodium borohydride. The formation of the stably cross-linked species with dL depends on the polymerase lysine 72 residue, which forms a Schiff base with the C-1 aldehyde during excision of an unmodified abasic site. In the case of a dL residue, attack on the lactone C-1 by lysine 72 proceeds more slowly and evidently produces an amide linkage, which resists further processing. Consequently dL residues may not be readily repaired by "short-patch" base excision repair but instead function as suicide substrates in the formation of protein-DNA cross-links that may require alternative modes of repair.