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.

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.

T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples.

In early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species of Borrelia spirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplify Borrelia DNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml for B. afzelii and 8 cells/ml for Borrelia burgdorferi and Borrelia garinii Clinical samples (n = 66) from confirmed or suspected early LD patients were also analyzed. B. burgdorferi was detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detected B. burgdorferi in blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) for Borrelia spp. in whole blood samples and is able to detect B. burgdorferi in clinical samples.

Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin.

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.

T2 magnetic resonance: a diagnostic platform for studying integrated hemostasis in whole blood--proof of concept.

BACKGROUND: Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1-25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling. METHODS: T2 magnetic resonance (T2MR) was used to directly measure integrated reactions in whole blood samples by resolving multiple water relaxation times from distinct sample microenvironments. Clotting, clot contraction, and fibrinolysis stimulated by thrombin or tissue plasminogen activator, respectively, were measured. T2MR signals of clotting samples were compared with images produced by scanning electron microscopy and with standard reference methods for the following parameters: hematocrit, prothrombin time, clot strength, and platelet activity. RESULTS: Application of T2MR methodology revealed conditions under which a unique T2MR signature appeared that corresponded with the formation of polyhedral erythrocytes, the dynamics and morphology of which are dependent on thrombin, fibrinogen, hematocrit, and platelet levels. We also showed that the T2MR platform can be used for precise and accurate measurements of hematocrit (%CV, 4.8%, R(2) = 0.95), clotting time (%CV, 3.5%, R(2) = 0.94), clot strength (R(2) = 0.95), and platelet function (93% agreement with light transmission aggregometry). CONCLUSIONS: This proof-of-concept study demonstrates that T2MR has the potential to provide rapid and sensitive identification of patients at risk for thrombosis or bleeding and to identify new biomarkers and therapeutic targets with a single, simple-to-employ analytic approach that may be suitable for routine use in both research and diverse clinical settings.

Relaxivity of gadolinium complexes detected by atomic magnetometry.

Laser atomic magnetometry is a portable and low-cost yet highly sensitive method for low magnetic field detection. In this work, the atomic magnetometer was used in a remote-detection geometry to measure the relaxivity of aqueous gadolinium-diethylenetriamine pentaacetic acid Gd(DTPA) at the Earth's magnetic field (40 µT). The measured relaxivity of 9.7±2.0 s(-1) mM(-1) is consistent with field-cycling experiments measured at slightly higher magnetic fields, but no cryogens or strong and homogeneous magnetic field were required for this experiment. The field-independent sensitivity of 80 fT Hz(-1/2) allowed an in vitro detection limit of ∼10 µM Gd(DTPA) to be measured in aqueous buffer solution. The low detection limit and enhanced relaxivity of Gd-containing complexes at Earth's field motivate continued development of atomic magnetometry toward medical applications.

Direct Testing for KPC-Mediated Carbapenem Resistance from Blood Samples Using a T2 Magnetic Resonance Based Assay.

Molecular-based carbapenem resistance testing in Gram-negative bacterial bloodstream infections (BSIs) is currently limited because of the reliance on positive blood culture (BC) samples. The T2Resistance™ panel may now allow the detection of carbapenemase- and other ß-lactamase encoding genes directly from blood samples. We detected carbapenem resistance genes in 11 (84.6%) of 13 samples from patients with BC-documented BSIs (10 caused by KPC-producing Klebsiellapneumoniae and 1 caused by VIM/CMY-producing Citrobacter freundii). Two samples that tested negative for carbapenem resistance genes were from patients with BC-documented BSIs caused by KPC-producing K. pneumoniae who were receiving effective antibiotic therapy. In conclusion, our findings suggest that the T2Resistance™ panel can be a reliable tool for diagnosing carbapenem-resistant Gram-negative bacterial BSIs.

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.

Sensitivity enhancement by exchange mediated magnetization transfer of the xenon biosensor signal.

Hyperpolarized xenon associated with ligand derivatized cryptophane-A cages has been developed as a NMR based biosensor. To optimize the detection sensitivity we describe use of xenon exchange between the caged and bulk dissolved xenon as an effective signal amplifier. This approach, somewhat analogous to 'remote detection' described recently, uses the chemical exchange to repeatedly transfer spectroscopic information from caged to bulk xenon, effectively integrating the caged signal. After an optimized integration period, the signal is read out by observation of the bulk magnetization. The spectrum of the caged xenon is reconstructed through use of a variable evolution period before transfer and Fourier analysis of the bulk signal as a function of the evolution time.

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt.

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.

T2MR and T2Candida: novel technology for the rapid diagnosis of candidemia and invasive candidiasis.

Candidemia and other forms of invasive candidiasis pose a significant diagnostic challenge. In order to provide the best treatment, it is important to accurately detect the fungal infection and identify the species. Historically, diagnosis of Candida infections depended upon three classical laboratory approaches: microbiologic, immunologic, histopathologic; and now includes new methods such as radiographic techniques, molecular, proteomic and biochemical methods. The T2Candida Panel has introduced a new class of infectious disease diagnostics that can rapidly detect and identify the causative pathogen of sepsis directly from a patient blood sample in a culture-independent manner. This test enables detection of Candida directly from the patient sample, a significant advance for the rapid and accurate diagnosis of invasive candidiasis.

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.

Distinguishing multiple chemotaxis Y protein conformations with laser-polarized 129Xe NMR.

The chemical shift of the (129)Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins. Here we show that the (129)Xe shift can sense more subtle changes: magnesium binding, BeF(3)(-) activation, and peptide binding by the Escherichia coli chemotaxis Y protein. (1)H-(15)N correlation spectroscopy and X-ray crystallography were used to identify two xenon-binding cavities in CheY that are primarily responsible for the shift changes. One site is near the active site, and the other is near the peptide binding site.

Kinetic studies of iron deposition in horse spleen ferritin using O2 as oxidant.

An optical flow cell provided a means to conveniently measure the rate of successive Fe(2+) oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe(2+) oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe(2+)/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)(3) formation and the presence (>95%) of Fe(OH)(3) deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5-50 Fe(2+)/HoSF established that the reaction is overall first order in Fe(2+) concentration. However, when full progress curves were analyzed at a variety of Fe(2+)/HoSF ratios, two first-order reactions (k(1) approximately 0.035 s(-1) and k(2) approximately 0.007 s(-1)) were found to contribute to the overall Fe(2+) oxidation reaction. The proportion of the fast reaction increased with increasing Fe(2+)/HoSF ratio until at approximately 400, it was the dominant reaction. For the Fe(2+)/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe(2+) oxidation. Comparison of successive additions of 1.0 Fe(2+)/HoSF showed that oxidation of the first 8-10 Fe(2+) produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H(+)/Fe(2+) ratio confirmed this difference in behavior by giving an H(+)/Fe(2+) ratio of approximately 1.0 below and 2.0 for ratios >30 Fe(2+)/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.

Kinetic studies of iron deposition in horse spleen ferritin using H2O2 and O2 as oxidants.

The reaction of horse spleen ferritin (HoSF) with Fe2+ at pH 6.5 and 7.5 using O2, H2O2 and 1:1 a mixture of both showed that the iron deposition reaction using H2O2 is approximately 20- to 50-fold faster than the reaction with O2 alone. When H2O2 was added during the iron deposition reaction initiated with O2 as oxidant, Fe2+ was preferentially oxidized by H2O2, consistent with the above kinetic measurements. Both the O2 and H2O2 reactions were well defined from 15 to 40 degrees C from which activation parameters were determined. The iron deposition reaction was also studied using O2 as oxidant in the presence and absence of catalase using both stopped-flow and pumped-flow measurements. The presence of catalase decreased the rate of iron deposition by approximately 1.5-fold, and gave slightly smaller absorbance changes than in its absence. From the rate constants for the O2 (0.044 s(-1)) and H2O2 (0.67 s(-1)) iron-deposition reactions at pH 7.5, simulations of steady-state H2O2 concentrations were computed to be 0.45 microM. This low value and reported Fe2+/O2 values of 2.0-2.5 are consistent with H2O2 rapidly reacting by an alternate but unidentified pathway involving a system component such as the protein shell or the mineral core as previously postulated [Biochemistry 22 (1983) 876; Biochemistry 40 (2001) 10832].

Applications of laser-polarized 129Xe to biomolecular assays.

The chemical shift sensitivity and significant signal enhancement afforded by laser-polarized 129Xe have motivated the application of 129Xe NMR to biological imaging and spectroscopy. Recent research done by our group has used laser-polarized 129Xe in biomolecular assays that detect ligand-binding events and distinguish protein conformations. The successful application of unfunctionalized and functionalized 129Xe NMR to in vitro biomolecular assays suggests the potential future use of a functionalized xenon biosensor for in vivo imaging.

T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood.

Candida spp. cause both local and disseminated infections in immunocompromised patients. Bloodstream infections of Candida spp., known as "candidemia," are associated with a high mortality rate (40%), which is mainly attributed to the long diagnostic time required by blood culture. We introduce a diagnostic platform based on T2 magnetic resonance (T2MR), which is capable of sensitive and rapid detection of fungal targets in whole blood. In our approach, blood-compatible polymerase chain reaction is followed by hybridization of the amplified pathogen DNA to capture probe-decorated nanoparticles. Hybridization yields nanoparticle microclusters that cause large changes in the sample's T2MR signal. With this T2MR-based method, Candida spp. can be detected directly in whole blood, thus eliminating the need for analyte purification. Using a small, portable T2MR detection device, we were able to rapidly, accurately, and reproducibly detect five Candida species within human whole blood with a limit of detection of 1 colony-forming unit/ml and a time to result of <3 hours. Spiked blood samples showed 98% positive agreement and 100% negative agreement between T2MR and blood culture. Additionally, performance of the assay was evaluated on 21 blinded clinical specimens collected serially. This study shows that the nanoparticle- and T2MR-based detection method is rapid and amenable to automation and offers clinicians the opportunity to detect and identify multiple human pathogens within hours of sample collection.