The T2Bacteria Assay Is a Sensitive and Rapid Detector of Bacteremia That Can Be Initiated in the Emergency Department and Has Potential to Favorably Influence Subsequent Therapy.

BACKGROUND: Bacteremia causes a major worldwide burden, in terms of financial and productivity costs, as well the morbidity and mortality it can ultimately cause. Proper treatment of bacteremia is a challenge because of the species-dependent response to antibiotics. The T2Bacteria Panel is a U.S. Food and Drug Administration-cleared and culture-independent assay for detection of bacteremia, including common ESKAPE pathogens-Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa-and provides species identification in as little as 3.6 h directly from blood. OBJECTIVE: Our aim was to evaluate the T2Bacteria assay performance and potential to affect patient care in the emergency department (ED). METHODS: ED patients from a Louisiana and Florida center were enrolled as part of the T2Bacteria Panel clinical study, which was prospective and noninterventional. Blood samples for blood culture (BC) and T2Bacteria were matched in time and anatomic location. RESULTS: Data from 137 ED patients were evaluated. Relative to BC, T2Bacteria showed 100% positive percent agreement and 98.4% negative percent agreement. In addition, for species on the T2Bacteria Panel, the T2Bacteria assay detected 25% more positives associated with infection, and on average identified the infectious species 56.6 h faster. The T2Bacteria assay covered 70.5% of all species detected by BC. Finally, relative to actual care, the T2Bacteria assay could have potentially focused therapy in 8 patients, reduced time to a species-directed therapy in 4 patients, and reduced time to effective therapy in 4 patients. CONCLUSIONS: In this ED population, the T2Bacteria assay was a rapid and sensitive detector of bacteremia from common ESKAPE pathogens and showed the theoretical potential to influence subsequent patient therapy, ranging from antibiotic de-escalation to faster time to effective therapy.

Magnetic Resonance-Based Diagnostics for Bleeding Assessment in Neonatal Cardiac Surgery.

PURPOSE: Infants undergoing a cardiac operation are at high risk for postsurgical bleeding. To date, there are no highly predictive models for postsurgical bleeding in this population. This study's objective was to assess the predictive ability of T2 magnetic resonance (T2MR). DESCRIPTION: T2MR uses magnetic resonance to detect clot formation characteristics on a small blood sample and provides hemostatic indicators that can assess bleeding risk. EVALUATION: This prospective, single-institution study enrolled 100 patients younger than 12 months old undergoing a cardiac operation from April 27, 2015, to September 21, 2016. The primary end point was postsurgical bleeding within 24 hours after the procedure. T2MR data were modeled with a binary recursive partitioning algorithm with randomized cross-validation. The tight clot metric produced the highest univariate discrimination of bleeding (receiver operator characteristic curve, 0.64; classification accuracy, 72%), and along with the platelet function metric, demonstrated highest relative importance based on Gini index splitting (Salford Systems, San Diego, CA). Multivariate modeling with cross-validation showed mean receiver operator characteristic curve area of 0.74 and classification accuracy of 82%. CONCLUSIONS: T2MR tight clot and platelet function metrics were associated with bleeding events.

ESKAPE Pathogens in Bloodstream Infections Are Associated With Higher Cost and Mortality but Can Be Predicted Using Diagnoses Upon Admission.

BACKGROUND: ESKAPE bacteria are thought to be especially resistant to antibiotics, and their resistance and prevalence in bloodstream infections are rising. Large studies are needed to better characterize the clinical impact of these bacteria and to develop algorithms that alert clinicians when patients are at high risk of an ESKAPE infection. METHODS: From a US data set of >1.1 M patient encounters, we evaluated if ESKAPE pathogens produced worse outcomes than non-ESKAPE pathogens and if an ESKAPE infection could be predicted using simple word group algorithms built from decision trees. RESULTS: We found that ESKAPE pathogens represented 42.2% of species isolated from bloodstream infections and, compared with non-ESKAPE pathogens, were associated with a 3.3-day increase in length of stay, a $5500 increase in cost of care, and a 2.1% absolute increase in mortality (P < 1e-99). ESKAPE pathogens were not universally more resistant to antibiotics, but only to select antibiotics (P < 5e-6), particularly against common empiric therapies. In addition, simple word group algorithms predicted ESKAPE pathogens with a positive predictive value of 7.9% to 56.2%, exceeding 4.8% by random guessing (P < 1e-99). CONCLUSIONS: Taken together, these data highlight the pathogenicity of ESKAPE bacteria, potential mechanisms of their pathogenicity, and the potential to predict ESKAPE infections upon admission. Implementing word group algorithms could enable earlier and targeted therapies against ESKAPE bacteria and thus reduce their burden on the health care system.

T2 Magnetic Resonance to Monitor Hemostasis.

There is a clinical need for pragmatic approaches to measure integrated hemostatic reactions in whole blood rapidly, using small volumes of blood. The authors have applied T2 magnetic resonance (T2MR) to assess coagulation reactions based on partitioning of red blood cells and proteins that occurs during fibrin formation and platelet-mediated clot contraction. T2MR is amenable to measuring clotting times, individual coagulation factors, and platelet function. T2MR also revealed a novel "hypercoagulable" signature characterized by fibrin clots almost insusceptible to fibrinolysis that surround tessellated arrays of polyhedral erythrocytes ("third peak"). This signature, which develops under conditions associated with intense clot formation in vitro, may help identify patients at risk of developing thrombosis and for monitoring antithrombotic therapies in the future.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance.

OBJECTIVES: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). METHODS: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. RESULTS: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. CONCLUSIONS: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests.

Embryonically inspired scaffolds regulate tenogenically differentiating cells.

Tendon injuries heal as scar tissue with significant dysfunction and propensity to re-injure, motivating efforts to develop stem cell-based therapies for tendon regeneration. For these therapies to succeed, effective cues to guide tenogenesis are needed. Our aim is to identify these cues within the embryonic tendon microenvironment. We recently demonstrated embryonic tendon elastic modulus increases during development and is substantially lower than in adult. Here, we examined how these embryonic mechanical properties influence tenogenically differentiating cells, by culturing embryonic tendon progenitor cells (TPCs) within alginate gel scaffolds fabricated with embryonic tendon mechanical properties. We showed that nano- and microscale moduli of RGD-functionalized alginate gels can be tailored to that of embryonic tendons by adjusting polymer concentration and crosslink density. These gels differentially regulated morphology of encapsulated TPCs as a function of initial elastic modulus. Additionally, higher initial elastic moduli elicited higher mRNA levels of scleraxis and collagen type XII but lower levels of collagen type I, whereas late tendon markers tenomodulin and collagen type III were unaffected. Our results demonstrate the potential to engineer scaffolds with embryonic mechanical properties and to use these scaffolds to regulate the behavior of tenogenically differentiating cells.

Actin cytoskeleton contributes to the elastic modulus of embryonic tendon during early development.

Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.

Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation.

Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated that mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus alternative means of assessing these properties are desirable. In this study, we examined lysyl oxidase (LOX)-mediated crosslinks as markers for mechanical properties during embryonic tendon formation and the potential to detect them non-destructively. We used tandem mass spectrometry (LC-MS/MS) to quantify changes in hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) crosslink density in embryonic chick tendon as a function of developmental stage. In addition, we assessed a multiphoton imaging approach that exploits the natural fluorescence of HP and LP. With both techniques, we quantified crosslink density in normal and LOX-inhibited tendons, and correlated measurements with mechanical properties. HP and LP crosslink density varied as a function of developmental stage, with HP-to-dry mass ratio correlating highly to elastic modulus, even when enzymatic crosslink formation was inhibited. Multiphoton optical imaging corroborated LC-MS/MS data, identifying significant reductions in crosslink density from LOX inhibition. Taken together, crosslink density may be useful as a marker of tissue mechanical properties that could be assessed with imaging non-destructively and perhaps non-invasively. These outcomes could have significant scientific and clinical implications, enabling continuous and long-term monitoring of mechanical properties of collagen-crosslinked tissues or engineered constructs.

Mechanical factors in embryonic tendon development: potential cues for stem cell tenogenesis.

Tendons are connective tissues required for motion and are frequently injured. Poor healing and inadequate return to normal tissue structure and mechanical function make tendon a prime candidate for tissue engineering; however functional tendons have yet to be engineered. The physical environment, from substrate stiffness to dynamic mechanical loading, may regulate tenogenic stem cell differentiation. Tissue stiffness and loading parameters derived from embryonic development may enhance tenogenic stem cell differentiation and tendon tissue formation. We highlight the current understanding of the mechanical environment experienced by embryonic tendons and how progenitor cells may sense and respond to physical inputs. We further discuss how mechanical factors have only recently been used to induce tenogenic fate in stem cells.

Characterization of mechanical and biochemical properties of developing embryonic tendon.

Tendons have uniquely high tensile strength, critical to their function to transfer force from muscle to bone. When injured, their innate healing response results in aberrant matrix organization and functional properties. Efforts to regenerate tendon are challenged by limited understanding of its normal development. Consequently, there are few known markers to assess tendon formation and parameters to design tissue engineering scaffolds. We profiled mechanical and biological properties of embryonic tendon and demonstrated functional properties of developing tendon are not wholly reflected by protein expression and tissue morphology. Using force volume-atomic force microscopy, we found that nano- and microscale tendon elastic moduli increase nonlinearly and become increasingly spatially heterogeneous during embryonic development. When we analyzed potential biochemical contributors to modulus, we found statistically significant but weak correlation between elastic modulus and collagen content, and no correlation with DNA or glycosaminoglycan content, indicating there are additional contributors to mechanical properties. To investigate collagen cross-linking as a potential contributor, we inhibited lysyl oxidase-mediated collagen cross-linking, which significantly reduced tendon elastic modulus without affecting collagen morphology or DNA, glycosaminoglycan, and collagen content. This suggests that lysyl oxidase-mediated cross-linking plays a significant role in the development of embryonic tendon functional properties and demonstrates that changes in cross-links alter mechanical properties without affecting matrix content and organization. Taken together, these data demonstrate the importance of functional markers to assess tendon development and provide a profile of tenogenic mechanical properties that may be implemented in tissue engineering scaffold design to mechanoregulate new tendon regeneration.

Novel strategies in tendon and ligament tissue engineering: Advanced biomaterials and regeneration motifs.

Tendon and ligaments have poor healing capacity and when injured often require surgical intervention. Tissue replacement via autografts and allografts are non-ideal strategies that can lead to future problems. As an alternative, scaffold-based tissue engineering strategies are being pursued. In this review, we describe design considerations and major recent advancements of scaffolds for tendon/ligament engineering. Specifically, we outline native tendon/ligament characteristics critical for design parameters and outcome measures, and introduce synthetic and naturally-derived biomaterials used in tendon/ligament scaffolds. We will describe applications of these biomaterials in advanced tendon/ligament engineering strategies including the utility of scaffold functionalization, cyclic strain, growth factors, and interface considerations. The goal of this review is to compile and interpret the important findings of recent tendon/ligament engineering research in an effort towards the advancement of regenerative strategies.

An improved murine femur fracture device for bone healing studies.

Murine models are commonly used to investigate bone healing and test new treatments before human trials. Our objective was to design an improved murine femur fracture device and determine optimal mass and velocity settings for maximal likelihood of transverse fracture. Fracture reproducibility was maximized using an adjustable kinetic energy level, a novel mouse positioning system and an electromagnet striker release assembly. Sixty wild-type mice of 8-12-week-old male and female with a weight of 26.4+/-6.1g were subjected to an experimental postmortem fracture in the left and right femur (n=120) using variable kinetic energy inputs. A best-fit prediction equation for transverse fracture was developed using multivariate linear regression. Transverse fracture was shown to correlate most highly with kinetic energy with a maximum likelihood at mv2=292 where m is mass (g) and v is velocity (m/s). Model validation with a group of 134 anesthetized C57BL/6 mice resulted in a favorable transverse fracture rate of 85.8%. Simple modifications to existing fracture devices can improve accuracy and reproducibility. The results may assist researchers studying the effects of genetic modifications and novel treatments on boney healing in murine femur fracture models. Maintaining kinetic energy parameters within suggested ranges may also aid in ensuring accuracy and reproducibility.