T2 Biosystems Research Articles

Induced Human Intestinal Organoids (iHIOs) as Model Systems for Chemotherapy-associated Clostridium difficile (CD) Infections

BackgroundPatients undergoing cytotoxic chemotherapy are ten times more likely to develop Clostridium difficile infections (CDI) than the general patient population. Efforts to outline pathophysiologic mechanisms underlying this disproportionate incidence have been limited by the lack of disease-representative experimental models. We hypothesized that iHIOs could serve as toxicity models to evaluate chemotherapy-associated CDIMethodsIntact iHIOs were exposed to cytotoxic chemotherapy (melphalan) in gut media at therapeutic doses (9 μg/mL; which is the equivalent of 140 mg/m2 human dose).Cellular death was assessed by accumulation of the membrane permeant dye, Sytox-orange added at 5-days post treatment. iHIOs were also exposed to CD toxin A and B (TcdA and TcdB respectively) and epithelial barrier damage assessed by actin mislocalization and loss of E-cadherin. For controls iHIOs were exposed / microinjected with saline/PBS. Morphological and histological changes were then captured using light and confocal microscopyResultsMorphologic and histologic assessments demonstrated cell death and epithelial barrier damageConclusioniHIOs demonstrate cell death on exposure to CD toxins and melphalan chemotherapy. These properties could be harnessed in establishing toxicity models for evaluation of chemotherapy-associated CDIDisclosures S. Apewokin, T2 biosystems: Investigator, Research support Astellas: Scientific Advisor, Consulting fee

Culture-Independent Molecular Methods for Detection of Antifungal Resistance Mechanisms and Fungal Identification.

Resistance to azoles and echinocandins has emerged as a significant factor affecting the clinical management of patients with invasive fungal infections. Immunosuppressed patients at high risk for invasive fungal infections often have prolonged or repeated exposure to antifungals resulting in either the well-documented selection of naturally occurring, less susceptible fungal species, or the in situ development of specific resistance mechanisms. Nucleic acid-based molecular diagnostics are particularly well suited for the rapid detection of low-abundance fungal pathogens and identification of the infecting pathogen to the genus and species levels, as well as assessment of resistance mechanisms. A wide range of molecular probing technologies involving real-time polymerase chain reaction (PCR) assays that facilitate direct analysis of a single infecting genome in a sterile blood specimen are available and have recently been commercialized (eg, Roche LightCycler SeptiFast and T2 Biosystems T2Candida). One of the exciting applications of molecular technology is the direct detection of specific resistance mechanisms that evolve during therapy. In principle, the detection of resistance mechanisms that have been independently validated to cause resistance provides a culture-independent biomarker for potential therapeutic failure. The emergence of real-time PCR assays utilizing allele-specific molecular detection technology that is highly sensitive, robust, and high-throughput has the potential to improve patient care by providing faster detection of drug-resistant infecting strains and to help inform therapeutic management.

Automated Detection of Candida auris Direct from Whole Blood by T2MR

Background Candida auris is now recognized worldwide as a virulent pathogen that is difficult to manage, resulting in high mortality rates. The majority of C.auris isolates have exhibited resistance to one or more antifungal agents. Nosocomial infections caused by C.auris are growing due to the increasing rate of colonization and environmental causes. The diagnostic tests available for the identification of C. auris are limited to date. Additionally, microbiological cultures and subsequent identification of Candida species require 2–5 days, and have a sensitivity of approximately 50%. Accurate diagnosis of a C. auris infection is also hampered by misidentification of C. auris as other species, commonly C. haemulonii and Saccharomyces cerevisiae.Here we evaluate the use of the T2MR platform for the highly sensitive, rapid species level identification of C. auris, C. lusitaniae and C. haemulonii in whole blood samples.MethodsA multiplex assay targeting C. auris, C. lusitaniae, and C. haemulonii was developed using cultured cells spiked in K2EDTA anticoagulated blood from healthy human donors. C. auris isolates received from the CDC were cultured overnight, automated cell counting was used to determine concentration. From this stock, the culture was diluted to a target titer, and inoculated into whole blood, followed by confirmation plating to confirm cell titer. Four mL spiked blood samples were processed on the T2Dx Instrument.ResultsSensitive and specific detection of C. auris was achieved direct from blood in less than 4 hours on the T2Dx Instrument. A Limit of Detection (LoD) for C. auris was demonstrated to be ≤10 CFU/mL. T2MR signals of samples spiked with target were approximately 30 times higher than samples with no target present, and no cross reactivity was observed between C.auris, C. haemulonii, C. lusitaniae and C. krusei.ConclusionLow concentrations of Candida cells can be detected and identified by T2MR. This prototype assay potentially allows for the rapid screening and identification of patients infected with Candida auris with high specificity and sensitivity, aiding in the hospital management and targeted therapy of this emerging multi-drug resistant pathogen.Disclosures B. Manning, T2 Biosystems: Employee and Shareholder, Salary; J. L. Snyder, T2 Biosystems: Employee and Shareholder, Salary; B. Chang, T2 Biosystems: Employee and Shareholder, Salary; C. Wong, T2 Biosystems: Employee and Shareholder, Salary; T. Higa, T2 Biosystems: Employee and Shareholder, Salary; R. Shivers, T2 Biosystems: Employee and Shareholder, Salary; T. J. Lowery, T2 Biosystems: Employee and Shareholder, Salary

Center-level variability in broad-spectrum antibiotic prescribing for children undergoing hematopoietic cell transplantion for acute leukemia.

BackgroundAntibiotic exposure after allogeneic hematopoietic cell transplant (HCT) is common. Exposure to specific classes of antibiotics after HCT has been associated with mortality, relapse and graft-vs.-host disease. Exploring differences in antibiotic utilization across hospitals could provide opportunities for comparative effectiveness studies and quality improvement interventions.MethodsWe conducted a retrospective cohort study of patients undergoing HCT for acute leukemia using a dataset merged from two sources: the Pediatric Health Information System and the Center for International Blood and Marrow Transplant Research. Medication use data were obtained from the day of transplant through engraftment. Hospital antibiotic utilization rates were reported as antibiotic days/1000 neutropenic days. Adjusted rates were calculated using a poisson regression controlling for age, sex, race, graft characteristics and days of ICU-level care.ResultsAfter adjustment, hospital rates of anti-pseudomonal antibiotic use varied from 410 to 1037 antibiotic days/1000 neutropenic days (Figure 1A) and for Gram-positive antibiotic use from 109 to 771 antibiotic days/1000 neutropenic days (Figure 1B). As shown in Figure 1, within anti-pseudomonal antibiotics, there was variation by hospital in the use of Fourth and 5th generation cephalosporins, anti-pseudomonal penicillins and carbapenems; variation in Gram-positive exposure was driven by vancomycin. Gram-positive antibiotic use was moderately associated with days of ICU-level of care (spearman correlation coefficient = .55) but anti-pseudomonal antibiotic use was not (Figure 2). There was no association between days of antibiotic exposure and 30-day mortality.ConclusionAmong a homogenous population of children undergoing transplantation for acute leukemia, both the volume and spectrum of antibiotic exposure in the immediate post-transplant period varied widely. These data present an opportunity for hospitals to benchmark their antibiotic utilization practices and can be further leveraged to assess the clinical impact of differential antibiotic exposure.Disclosures B. T. Fisher, Pfizer, Inc.: Grant Investigator, Research support. Merck, Inc.: Investigator, Research support. T2 Biosystems, Inc.: Investigator, Research support. Ansun Biopharma: Investigator, Research support

Hemophilia a Clots Generated with Recombinant Factor VIIa Differed in Structure and Composition from Those Formed with Factor VIII

Patients with severe factor VIII (FVIII) deficiency (hemophilia A [HemA]) develop neutralizing antibodies (inhibitors) against FVIII in up to ~30% of cases. For HemA patients with inhibitors, activated recombinant factor VII (rFVIIa) is a treatment option. High levels of rFVIIa are required for treating HemA patients with inhibitors to induce direct activation of factor X on the surface of activated platelets via a tissue factor (TF)-independent mechanism (Hoffman M, Monroe DM. Thromb Res. 2010;125(suppl 1):S16-S18). To assess how rFVIIa-mediated clot formation in HemA patients with inhibitors may differ from unaffected individuals, we compared the effect of rFVIIa on HemA versus control (or HemA supplemented with 100% FVIII) clot formation in human and/or mouse systems. By TF-induced thrombin generation assay, increasing rFVIIa from 5 nM to 100 nM did not appreciably alter the kinetics or extent of thrombin generation compared with the same human HemA plasma containing 100% FVIII. Confocal microscopy of human HemA plasma clots generated with 75 nM rFVIIa and TF showed few branching fibrin fibers and an open fibrin meshwork. In contrast, TF-induced coagulation of the same HemA plasma containing 100% FVIII formed fibrin clots with numerous branches, interconnecting to form a dense meshwork. To confirm that these findings reflect rFVIIa-mediated clot formation in vivo, we assessed the intrinsic coagulation of mouse HemA whole blood collected without anticoagulant and spiked with rFVIIa. Intrinsic coagulation with rFVIIa was assessed by T2 magnetic resonance (T2MR), a technique capable of monitoring the separation of whole blood into serum, loose-clot, and tight-clot compartments during coagulation (Skewis et al. Clin Chem. 2014;60:1174-1182; Cines et al. Blood. 2014;123:1596-1603). By T2MR, rFVIIa induced the separation of HemA whole blood into the serum and clot compartments, indicating that the reduced fibrin generation with rFVIIa did not interfere with whole blood coagulation. Furthermore, saphenous vein puncture of HemA mice treated with rFVIIa showed a dose-dependent decrease in clot times. Scanning electron microscopy of the clots extracted from these HemA mice indicated markedly different composition than clots extracted from wild-type mice. In wild-type clots, fibrin and polyhedral erythrocytes formed a large proportion of the total structures. In contrast, clots from rFVIIa-treated HemA mice consisted primarily of platelets and erythrocytes with forms intermediate between discoid and polyhedral but, surprisingly, low fibrin content. Taken together, these data suggest that rFVIIa-mediated clot formation may require greater activated platelet involvement, which would be consistent with the TF-independent mechanism of action proposed for rFVIIa in HemA. Finally, the compositional difference between clots from wild-type versus HemA mice dosed with rFVIIa suggest that evaluating HemA therapies for their ability to form more physiologic clots could be an approach to improve treatment options for patients with HemA. DisclosuresLeong:Bayer: Employment. Xu:Bayer: Employment. Mallari:Bayer: Employment. Wong:Bayer: Employment. Sim:Bayer: Employment. Cuker:Stago: Consultancy; Genzyme: Consultancy; Amgen: Consultancy; Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding. Marturano:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment. Kauser:Bayer: Employment. Weisel:Bayer: Research Funding.

Characterization of Bleeding in Hemophilia Carriers and Comparison to Women with Type 1 Von Willebrand Disease, Type 3 Von Willebrand Disease Obligate Carriers and Controls

Background: Hemophilia carriers report abnormal bleeding, even when factor VIII or IX levels are normal. Information comparing bleeding events between carriers and women with other inherited bleeding disorders is lacking. Purpose: The purpose of our study was to characterize bleeding in hemophilia carriers using the International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) and to compare it with bleeding in normal controls, women with Type 1 VWD and Type 3 VWD obligate carriers (OC). Method: This was a prospective, observational, cross-sectional study performed by members of GEHEP (Global Emerging HEmostasis Panel). Study participants were recruited from GEHEP members9 clinics in North America (Kingston, Canada, Detroit and Philadelphia, USA), Europe (Malaga, Spain; Milan, Italy; Munich, Germany; Oslo, Norway) and South Africa (Johannesburg). Potential participants were identified through local patient databases and approached during clinic visits. All participants signed informed consent. Hemophilia carriers were defined by a documented FVIII or FIX mutation and/or by an appropriate family history (daughter of a man with hemophilia or mother of two sons with hemophilia or mother of one son with hemophilia with at least one other affected male relative). Demographic information was collected using a CRF and the ISTH-BAT was completed for each participant by study personnel. Existing ISTH-BAT data for women with Type 1 VWD, Type 3 VWD OC and age-matched female controls were used for comparison. Results: A total of 329 participants were included in this study; 168 hemophilia carriers, 83 women with Type 1 VWD, 32 Type 3 VWD OC and 46 female normal controls. Hemophilia carriers and normal controls were similar in age (40.1 vs 41.6, p=0.445). The mean overall ISTH-BAT bleeding score (BS) was significantly higher in carriers than in controls (5.7 vs 2.48, p Conclusion: In summary, our study showed that hemophilia carriers report significantly more bleeding by overall ISTH-BAT BS than age-matched female controls. Carriers experience both mucocutaneous bleeding as well as musculoskeletal bleeding. They score higher for mucocutaneous bleeding when compared with controls and when compared with Type 3 VWD OC. Overall Type 1 VWD patients experience more severe mucocutaneous bleeding than hemophilia carriers. However, hemophilia carriers report more musculoskeletal bleeding in the form of hemarthrosis and hematomas than all other groups. A comparison of overall ISTH-BAT BS between groups shows that bleeding in women with Type 1 VWD > hemophilia carriers > Type 3 VWD OC > controls. Additional research into the underlying pathophysiology of this abnormal bleeding is a critical next step in understanding and determining how to appropriately manage these patients. Disclosures Bidlingmaier:Novo Nordisk: Honoraria; Sobi: Honoraria; Pfizer: Honoraria; Biotest: Honoraria; Baxalta: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria, Research Funding. Mingot-Castellano:Amgen: Consultancy; Pfizer: Consultancy; Novo Nordisk: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; Novartis: Consultancy; Bayer: Consultancy, Research Funding. Chitlur:Novo Nordisk: Consultancy; Baxalta: Honoraria; Bayer: Honoraria; Biogen-Idec: Honoraria; Pfizer: Honoraria. Fogarty:Bayer Healthcare: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Baxter/Baxalta: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Biogen: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity9s Board of Directors or advisory committees; CSL Behring: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity9s Board of Directors or advisory committees; Pfizer: Employment, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Research Funding. Cuker:T2 Biosystems: Research Funding; Genzyme: Consultancy; Biogen-Idec: Consultancy, Research Funding; Amgen: Consultancy; Stago: Consultancy. Mancuso:Bayer Healthcare: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Baxalta: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; Sobi/Biogen Idec: Consultancy, Speakers Bureau; Pfizer: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Kedrion: Consultancy. Holme:Baxalta, now part of Shire: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Investigator Clinical Studies. Mathew:Bayer: Employment. James:CSL Behring: Research Funding; Octapharma: Research Funding; Biogen: Consultancy; Basalt: Consultancy; Bayer: Research Funding.

Spk-9001: Adeno-Associated Virus Mediated Gene Transfer for Hemophilia B Achieves Sustained Mean Factor IX Activity Levels of >30% without Immunosuppression

▪Background: Earlier data demonstrated long-term expression of factor IX (mean FIX:C ~5.1%) following AAV8-mediated gene transfer at 2 x1012 vg/kg in hemophilia B (Nathwani et al., 2014). While the clinical improvement imparted by stable FIX levels is clear, these levels of expression fall short of trough values obtained by long-acting FIX prophylaxis (Santagostino et al. 2016), and of natural history data suggesting that levels of ~12% are required to eliminate spontaneous hemarthroses (den Uijl et al. 2011). Achieving higher levels of FIX:C with dose escalation has not been possible without eliciting a dose-dependent, capsid-specific immune response that may prevent sustained expression and efficacy (Mingozzi et al. 2007, Monahan et al. 2015). We sought to develop a highly efficient vector capsid and expression cassette that could be administered at low doses to achieve hemostatic FIX expression without need for immunosuppression.Methods: The investigational product, SPK-9001, utilizes a bioengineered AAV capsid (Spark100) with liver specific tropism. The prevalence of neutralizing antibodies (NAb) to Spark100 among sampled hemophilia B sera was 40% (Anguela et al. 2015). The expression cassette is a codon-optimized, single-stranded transgene encoding FIX Padua, a naturally occurring variant with a single amino acid substitution (R338L) that confers ~8-fold greater specific activity compared to wild-type FIX (Simioni et al. 2009). Data on bleeding and factor infusions in the year prior to enrollment were retrospectively compiled. Laboratory values, bleeding frequency, FIX consumption, changes in activity and quality-of-life via Haem-A-QoL were prospectively evaluated after vector infusion.Results: We enrolled 9 subjects, of whom 2 failed screening for liver fibrosis and 7 were infused with SPK-9001 at a dose of 5 x1011 vg/kg. Infused subjects were adult males ages 18-52 years with baseline FIX:C </=2% and Spark100 NAb titer of <1:1 or 1:1. Table 1 outlines infused subject data with a follow up interval of >2-34 weeks after vector infusion. Figure 1 outlines subject vector-derived FIX:C for the first 12 weeks. There have been no vector or procedure related adverse events. Steady-state FIX expression is reached by 12 weeks after vector infusion, resulting in a mean FIX:C of 32.3% ±6.5%. To date, no subjects required immunosuppression or demonstrated evidence of a cytotoxic immune response (characterized by loss of FIX activity, elevation of transaminase values >/=1.5-times the upper limit of normal, and positive IFN-gammaELISPOT response to capsid peptides). No subjects developed a FIX inhibitor or demonstrated ELISPOT reactivity to the FIX (R338L) gene product. Subject 3 infused with FIX concentrate for a suspected ankle bleed 2 days after vector infusion. Beyond this, no subjects required factor or experienced any bleeding events. The 4 subjects previously maintained on prophylaxis safely stopped without break-through bleeding. As of today (cumulative 724 days post vector infusion), total factor consumption was reduced by 543,589 IU, tantamount to a cumulative savings of $1,182,298 USD.Six of 7 subjects report increased physical activity and improved quality of life.Conclusion: As of 8/4/2016, we report the highest and most consistent levels of sustained vector-derived FIX:C following FIX gene transfer. Levels of FIX:C achieved by SPK-9001 permitted termination of prophylaxis, prevention of bleeding, and nearly complete cessation of factor use. Despite the heterogeneity in subjects with respect to presence and extent of hemophilic arthropathy, age, and co-morbidities, consistency of transgene expression and clinical outcomes have been observed in all participants studied to date. A vector dose of 5x1011 vg/kg is the lowest dose currently reported in hemophilia gene transfer trials; the absence of any observed CD8+ T cell immune response supports the hypothesis that lowering the dose can reduce or eliminate the risk of a capsid-specific immune response and maximize efficacy. In summary, preliminary data suggest SPK-9001 safely and consistently produces sustained elevation in FIX:C levels sufficient to prevent spontaneous hemarthroses without the need for factor consumption or immunosuppression. [Display omitted] [Display omitted] DisclosuresDucore:Octapharama: Membership on an entity’s Board of Directors or advisory committees; LFB: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees; Baxalta (Shire): Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees. Cuker:Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding; Genzyme: Consultancy; Stago: Consultancy; Amgen: Consultancy. McGuinn:Spark: Research Funding; Biogen: Research Funding; Novo Nordisk: Research Funding; Baxalta: Research Funding. Luk:Spark Therapeutics, Inc.: Employment. Wright:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001. Chen:Spark Therapeutics, Inc.: Employment. Hui:Spark Therapeutics, Inc.: Employment. Wachtel:Spark Therapeutics, Inc.: Employment. Urich:Spark Therapeutics, Inc.: Employment. Takefman:Spark Therapeutics, Inc.: Employment. Couto:Spark Therapeutics, Inc.: Employment. Carr:Pfizer, Inc.: Research Funding. Anguela:Spark Therapeutics, Inc.: Employment, Patents & Royalties: SPK-9001. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001.

Canon Gets 20% Equity Stake in T2 Biosystems

Clinical OMICsVol. 3, No. 10 DIAGNOSTICSCanon Gets 20% Equity Stake in T2 BiosystemsPublished Online:27 Oct 2016https://doi.org/10.1089/clinomi.03.10.18AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Canon Gets 20% Equity Stake in T2 Biosystems." , 3(10), p. 20FiguresReferencesRelatedDetails Volume 3Issue 10Nov 2016 InformationCopyright © GEN PublishingTo cite this article:Canon Gets 20% Equity Stake in T2 Biosystems.Clinical OMICs.Nov 2016.20-20.http://doi.org/10.1089/clinomi.03.10.18Published in Volume: 3 Issue 10: October 27, 2016PDF download

Canon invests in T2 Biosystems

Canon USA has purchased a nearly 20% stake in T2 Biosystems, a maker of time-domain nuclear magnetic resonance diagnostics, for $40 million. T2 has worked with the molecular diagnostics arm of Canon since 2015 to develop a test panel for Lyme disease. T2 expects to use the funds to grow its business and launch a sepsis detection panel in 2017. The firm’s technology was developed by scientists at Massachusetts General Hospital and MIT.

Implants for Cost-Effective and Accessible Treatment of Gynecologic and Gastrointestinal Cancer Metastasis by Local and Sustained Low-Dose Chemotherapy

Abstract 46 Gynecologic and gastrointestinal malignancies are two of the most prevalent cancer types in low and middle- income countries (LMICs), affecting more than 2 million individuals and killing more than 1 million patients annually. These malignancies at an advanced stage metastasize locally but extensively; this spread is a primary cause of morbidity and mortality, affecting 60-80% of patients in LMICs. Localized chemotherapy can benefit survival of patients with such metastases. Localized chemotherapy is, however, essentially unattainable in resource-limited settings, even though many chemotherapy agents are on the WHO list of essential medicines and are currently off patent. This is because of the high cost of, and frequent hospitalizations required under, the current regimen, as well as the morbidity of bolus dosing. Continuous, low-dose chemotherapy via a locally implanted device can address these adoption barriers. Previous attempts to create implants for localized chemotherapy delivery have been hindered by poorly controlled drug release and inhibiting form factors. This work explores the development of a nonresorbable and laparoscopically deployable implant to administer continuous low-dose chemotherapy. The feasibility of chemotherapy using the proposed implant in LMICs is assessed using physician interviews and literature reviews. Tissue-like silicone elastomers are used to create a matrix-type drug delivery implant that minimizes soft tissue irritation and risk of rupture while allowing laparoscopic manipulation. The synthesis and drug release profile of the silicone-based matrix are characterized for small hydrophilic active agents. Configurations allowing implant deployment through laparoscopic instruments are explored. Proof-of-concept controlled release of a hydrophilic small molecule from a lipophilic, tissue-like silicone elastomer that can be scaled to human form factors is thus established. Localized, low-dose chemotherapy delivered via a fully implantable device holds promise to dramatically reduce the cost and resources necessary for treating advanced-stage gynecologic and gastrointestinal malignancies in LMICs, improving cancer patient outcomes in resource-limited settings. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST: Aikaterini Mantzavinou No relationship to disclose Michael J. Cima Leadership: T2 Biosystems, MicroChips Biotechnologies, Taris BioMedical Stock or Other Ownership: T2 Biosystems, MicroChips Biotechnologies, Taris BioMedical Research Funding: Pfizer Patents, Royalties, Other Intellectual Property: Too many to describe (over 50 patents) Expert Testimony: Apotex Laura Melanie Tanenbaum Patents, Royalties, Other Intellectual Property: System and Method for Sterile Sheathing of a Medical Probe

T2Candida Provides Rapid and Accurate Species Identification in Pediatric Cases of Candidemia.

The goal of this study is to assess the ability of the T2Candida platform (T2 Biosystems, Lexington, MA) to accurately identify Candida species from pediatric blood specimens with low volumes. Whole blood from 15 children with candidemia was collected immediately following blood culture draw. The amount of blood required by the system was reduced by pipetting whole blood directly onto the T2Candida cartridge. Specimens were subsequently run on the T2Dx Instrument (T2 Biosystems). The T2Candida panel provided the appropriate result for each specimen compared with blood culture-based species identification and correctly identified 15 positive and nine negative results in 3 to 5 hours. While the time to species identification for blood culture was not reported, the T2Candida results include species data. T2Candida can be used to efficiently diagnose or rule out candidemia using low-volume blood specimens from pediatric patients. This could result in improved time to appropriate antifungal therapy or reduction in unnecessary empirical antifungal therapy.

A Blood Scan for Sepsis?

Each day, we carry up to 6 pounds of microorganisms around inside our bodies. Most of the time, this microbiome is kept in check by our immune system. But age, surgery, or chronic disease can weaken our immune system, giving these microorganisms a chance to gain hold. Under the right (or wrong) conditions, this microbial coup (or bad foreign bugs) can cause sepsis. Each year, sepsis strikes 1 million Americans, and can kill up to half of those patients. That's more than AIDS, prostate cancer, and breast cancer combined. Mortality is reduced when the cause of the infection—the specific species of bacteria or fungus—is quickly identified and targeted medications are given. “For every hour sepsis goes undiagnosed, mortality increases by eight percent,” says scientist Tom Lowery. “Differences in time of identification could impact the mortality of patients.” Yet today's gold standard sepsis tests can take 2–5 days to identify the basis for infection. Enter Lowery and the team at T2 Biosystems, a company that is using patented nanotechnology-based T2 magnetic resonance (T2MR) for the “detection of pathogens, biomarkers, and other abnormalities in a variety of unpurified patient sample types.” Lowery is their chief scientific officer. In September 2014, the US Food and Drug Administration approved the company's first technology: the T2Candida Panel, a test that promises to identify, within hours, fungal infections that can cause sepsis (1). Here, we consider T2Candida and its potential in the laboratory. The T2Candida Panel is a nonoptical, non–culture-based …

A Cross-Sectional Analysis of Cardiovascular Disease in the Hemophilia Population

A Cross-Sectional Analysis of Cardiovascular Disease in the Hemophilia Population

Inappropriate Listing of Heparin As an Allergy in the Electronic Medical Record Due to Misdiagnosis of Heparin-Induced Thrombocytopenia: Frequency and Consequences

Introduction Heparin-induced thrombocytopenia (HIT) is a prothrombotic complication of heparin therapy. Treatment involves discontinuation of heparin and initiation of an alternative anticoagulant. Misdiagnosis is common and may result in unnecessary exposure of thrombocytopenic patients to costly direct thrombin inhibitors and their approximate 1% daily risk of major hemorrhage. Another potential and previously unstudied consequence of misdiagnosis is the inappropriate listing of heparin as an allergy in the electronic medical record (EMR). We hypothesized that inappropriate listing of heparin as an allergy due to misdiagnosis of HIT is relatively common, often persists beyond the index hospitalization, and is associated with unnecessary avoidance of heparin as well as increased bleeding and costs. Methods We conducted a retrospective cohort study of patients with an inappropriate heparin allergy listed in the EMR due to misdiagnosis of HIT. We searched the EMR of a tertiary care center and a community hospital in our health system for patients with a new heparin allergy documented between 2004 and 2011. Patients were eligible for the study if the reason for allergy listing was suspicion for acute HIT and laboratory testing for HIT had been performed within 60 days of the allergy listing. We excluded patients that were listed as having a heparin allergy due to a remote history of suspected HIT, an adverse reaction to heparin other than HIT, or if the allergy was listed for an unknown reason. Charts of eligible subjects were reviewed and demographic, clinical, and laboratory data were extracted using a standardized data collection form. Subjects were defined as "negative for HIT" if they had a 4T score ≤3 or negative laboratory testing for HIT (ELISA &lt;1.0 optical density units and negative serotonin release assay). All other subjects were considered to have "possible HIT." Results Among 903 patients with a new allergy to heparin documented during the time period of interest, 239 were eligible for inclusion in the study. Of these 239 subjects, 100 (42%) met the prespecified definition of "negative for HIT" (15 had a 4T score ≤3, 49 had negative laboratory testing, and 36 had both a 4T score ≤3 and negative laboratory testing) . Sixty-eight of the subjects who were negative for HIT (68%) received an alternative parenteral anticoagulant during the index admission: 52 received argatroban, 18 lepirudin, 2 both argatroban and lepirudin. No patients were treated with fondaparinux or bivalirudin. Median length of time on an alternative parenteral anticoagulant was 10.5 days. Among the 68 patients who received unnecessary argatroban and/or lepirudin, 45 (66%) met ISTH criteria for major bleeding. The majority of these subjects met criteria for a major bleed based on a decrease in hemoglobin of at least 2.0 g/dL (n=40, 58.8%) and/or transfusion of at least 2 units of packed red blood cells (n=33, 48.5%). There were 2 subjects who had a symptomatic bleed in a critical area or organ. No subjects had a fatal hemorrhage. Seventy-five (75%) of the 100 subjects that were negative for HIT had an inappropriate allergy to heparin listed in the EMR at the time of discharge from the index hospitalization, and 68 (68%) had an inappropriate allergy to heparin that remained in effect as of August 2015. These 68 patients have had 68 subsequent hospitalizations within our health system after the index admission. Conclusions We found that a substantial percentage (42%) of patients with a heparin allergy documented in the EMR due to suspected HIT were clearly negative for HIT based on review of clinical and laboratory data. Many of these patients were unnecessarily treated with a direct thrombin inhibitor (68%) and experienced major bleeding (66%). Inappropriate heparin allergy listing persisted in the EMR in most patients (75%) beyond the index hospitalization, suggesting that heparin may be unnecessarily avoided and alternative parenteral anticoagulants used in subsequent admissions. Disclosures Cuker: Bracco: Consultancy; CSL Behring: Consultancy; Genzyme: Consultancy; T2 Biosystems: Research Funding.

An NMR Renaissance

In the field of nuclear magnetic resonance (NMR) spectroscopy, bigger and more expensive have always meant better. Although that maxim is still true, a new generation of companies contends that smaller benchtop or even handheld NMRs can do much of the yeoman’s work of identifying molecular structures that room-sized NMRs do. Makers of the smaller tools—firms such as Magritek; Thermo Fisher Scientific, which purchased start-up picoSpin in 2012; Nanalysis; T2 Biosystems; and WaveGuide—don’t expect their modestly priced machines will replace expensive high-field instruments tricked out with cryo-cooled magnets and separate operator consoles. But aided by modern electronics, improvements in permanent magnet design, and advances in software, compact-NMR makers say their small tools will become more commonplace over the next five years. What’s more, makers of smaller NMRs say their tools will be accessible to more than just scientists skilled at using the traditional machines. Expect to see the new instruments, ...

Miniaturized T2MR Magnetic Resonance System for Analysis of Hemostasis and Detection of Impaired and Prothrombotic Blood Disorders

AbstractAbstract 1118There are no currently available tests to rapidly assess and reliably identify both impaired hemostasis and hypercoagulable states, in part because of difficulties in measuring integrated reactions in whole blood using a single sensitive and clinically useful platform. Methods like T2 magnetic resonance (T2MR) can provide rich information from complex samples, such as changes in the blood during hemostasis, by measuring signals emanating from the hydrogen nuclei within the sample, primarily in water. We used a 14”x6”x7” portable instrument to measure changes in T2MR that provide a continuous report on the dynamically changing microscopic environment of water during coagulation of whole blood (WB), platelet-poor or platelet-rich plasma (PRP).In these initial foundational studies, we measured T2MR continuously over 20 mins using 34 μL blood samples from consented normal adult donors. We tested clotting of WB initiated by the addition of thrombin or kaolin + calcium. Platelet activation was achieved in WB by addition of ADP or arachidonic acid in the presence of reptilase and factor XIIIa with and without addition of the platelet inhibitors aspirin or 2-methylthioadenosine 5’-monophosphate (2-MeSAMP) and results were confirmed by standard platelet aggregometry. At normal platelet counts from 1.5–3×105/μL and normal hematocrit (HCT) from 38%-48%, T2MR gave two curves corresponding to: (1) water trapped within a retracted clot and (2) water in the surrounding liquid, i.e. serum (Fig. 1a). Platelet counts <1×105/μL or addition of aspirin or 2-MeSAMP eliminated the signal originating from clot retraction (Fig. 1b). High platelet counts (3–13×105/μL) and high thrombin concentrations (10 u/mL) led to a third, even more highly ordered signal (Fig. 1c). The signal also revealed the influence of hematocrit on clot formation/retraction over a wide range of values (20%-73%). Platelet inhibition by aspirin or 2-MeSAMP decreased or eliminated the signal originating from clot retraction (Fig. 1d).Our proof-of-principle studies show that T2MR technology can be applied to measurement of blood clotting across a range of hemostatic conditions. This single technology may be applicable to the study, diagnosis, and management of a spectrum of disorders that range from impaired hemostasis to hypercoagulable states. These T2MR studies represent the first application of this technology to hemostasis and thrombosis. Additional studies will be needed to develop a more complete understanding of the biochemical events measured by T2MR and to more fully explore its clinical utility. [Display omitted] Disclosures:Cines:T2 Biosystems: Research Funding. Lebedeva:T2 Biosystems: Research Funding. Massefski:T2 Biosystems: Employment. Papkov:T2 Biosystems: Employment. Thayer:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment.

Novel Diagnostic Assays for Heparin-Induced Thrombocytopenia

AbstractAbstract 267 IntroductionHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by platelet, monocyte, and endothelial cell-activating antibodies (Abs) against ultralarge complexes of platelet factor 4 (PF4) and heparin. Laboratory testing plays a key role in the diagnosis of HIT, but is associated with important shortcomings. Immunoassays such as the PF4/heparin ELISA frequently yield false-positive results due to their inability to discriminate cellular activating-Abs from their non-pathogenic counterparts. Functional assays such as the 14C-serotonin release assay (SRA) are more specific, but are unfeasible for most clinical laboratories due to the requirement for radioisotope and fresh platelets from reactive donors.KKO is a monoclonal Ab that causes a HIT-like thrombocytopenic disorder in a mouse model. Binding of KKO to immobilized PF4/heparin is inhibited by human HIT plasma, but not by plasma from patients with non-pathogenic anti-PF4/heparin Abs.1 We exploited this property of KKO to develop a KKO-inhibition (KKO-I) ELISA to detect platelet-activating Abs. We recently described a system to measure cell activation by HIT Abs: DT40 (chicken B lymphocyte) cells transfected with human FcgRIIa coupled to a luciferase reporter.2 We hypothesized that this system (DT40-luc) could be used to identify cell-activating anti-PF4/heparin Abs without need for donor platelets or radioactivity. Here we describe the KKO-I and DT40-luc assays and compare their performance to two commercially available immunoassays and the SRA in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin Abs. MethodsPatient samples consecutively referred to a clinical coagulation laboratory for HIT laboratory testing that tested positive by polyspecific anti-PF4/heparin ELISA were included. In addition to the polyspecific ELISA, citrated plasma samples from all patients were tested by an IgG-specific PF4/heparin ELISA, an in-house SRA, and the investigational KKO-I and DT40-luc assays. A 4Ts score to estimate the clinical likelihood of HIT was determined for each subject. The investigator performing 4Ts scoring was blinded to the results of HIT laboratory assays. Investigators performing the KKO-I and DT40-luc assays were blinded to the 4Ts score and the results of the SRA and anti-PF4/heparin ELISA.The KKO-I and DT40-luc assays were performed as previously described.1,2 HIT was defined as the combination of an intermediate or high probability 4Ts score (≥4) and a positive SRA. The performance of each assay with respect to this reference standard was evaluated by receiver-operating characteristic (ROC) analysis. Areas under the ROC curves (AUCs) were calculated and compared using the Delong method for correlated samples. ResultsFifty-eight subjects were enrolled, 21 of whom met prespecified criteria for HIT. There were no significant differences in demographic characteristics between the 21 HIT-positive and 37 HIT-negative subjects.The ability of the polyspecific ELISA, IgG-specific ELISA, KKO-I, and DT40-luc assay to discriminate HIT-positive from HIT-negative subjects is shown in Figure 1. HIT-positive plasma showed significantly greater mean inhibition of KKO binding than HIT-negative plasma (70.1%, 95% CI 64.8–75.4 vs. 40.4%, 33.5–47.4, p<0.0001) (Figure 1C). Plasma from HIT-positive subjects also induced significantly greater luciferase activity (3.14, 2.25–4.03 vs. 0.96, 0.85–1.07, p<0.0001) in the DT40-luc assay (Figure 1D).ROC curves for each assay are shown in Figure 2. The AUC for KKO-I (0.92, 0.85–1.00) was significantly greater than the AUC for the polyspecific (0.82, 0.70–0.95) and IgG-specific (0.76, 0.62–0.90) ELISAs (p<0.05 for both comparisons). The AUC for DT40-luc (0.89, 0.79–0.99) was significantly greater than the AUC for the IgG-specific (p=0.046), but not the polyspecific ELISA (p=0.28). ConclusionKKO-I and DT40-luc showed better discrimination than commercially available ELISAs in a small cohort of patients with suspected HIT and anti-PF4/heparin Abs. These assays are simple to perform, do not require donor platelets or radioactivity, and hold promise for improving the specificity and feasibility of HIT laboratory testing. Further evaluation in a larger cohort of patients is required. [Display omitted] [Display omitted] Disclosures:Cuker:Baxter: Consultancy, Research Funding; Bayer: Consultancy; Canyon: Consultancy; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Stago: Research Funding. Arepally:Teva Pharmaceuticals: Research Funding. Cines:Amgen: Consultancy; GSK: Consultancy; Eisai: Consultancy; T2 Biosystems: Research Funding.

Higher Infused Cell Dose Is Associated with a Decreased Risk of Invasive Fungal Infections After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 4134Invasive fungal infections/disease (IFI/IFD) are a major cause of morbidity and mortality after hematopoietic stem cell transplants (HCT), but their incidence and risk factors are not well defined. We performed a retrospective review of 270 consecutive adults (152 M/118 F), median age 53 (18–75) years, with hematologic malignancy receiving allogeneic HCT at Massachusetts General Hospital between 2000 to 2010 to determine the incidence, risk factors and outcomes of patients that developed IFD. All patients received antifungal prophylaxis from the start of conditioning until Day +100; 90% received fluconazole 400 mg daily. We defined IFD using the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group definitions, defining proven cases as evidence of fungal elements in a sterile tissue, and probable as presence of fungal elements in non sterile tissue or positive Galactomannan antigen or β-D-Glucan test in patients with clinical features suggestive of IFI. Diagnoses were Acute Myeloid Leukemia (34%), Non Hodgkin’s Lymphoma (24%), Myelodysplastic syndrome (15%), Acute Lymphoblastic Leukemia (8%), Chronic Lymphocytic Leukemia (5%), Myeloproliferative disorders, Hodgkin Lymphoma and Multiple Myeloma (4% each), and others (2%). 15% had a prior autologous transplant. Conditioning included reduced intensity (64%) or myeloablative (36%) regimens. Donor source was fully HLA matched (MRD) in 60%, matched unrelated (MUD) in 20%, Haploidentical in 12% and Cord Blood in 8%. 41% received ATG based aGVHD prophylaxis vs non ATG based in 59%. 11% were diabetic, while 6% had a history of IFD prior to transplant. 55% received systemic steroids for GVHD and 34% received TPN during transplant course. Median follow up was 28 (1.0–141.0) months. 49 distinct episodes of IFI were observed in 40 subjects (15%). Aspergillus spp (47%) was most frequently encountered, followed by Candida spp (43%). Mucor was isolated in 1 case. Among the Candida spp, the majority (52%) were non albicans- Candida glabrata (8) vs 1 each of Candida krusei, Candida tropicalis and Candida parapsilosis. Most frequently infected site was the lungs (69%), followed by the blood stream and paranasal sinuses (14% each), and GI tract (4%). Spleen (hepatosplenic candidiasis), kidneys and brain were affected in 1 patient each. In 6%, involvement of both the lungs and sinuses was observed. The estimated probability of early IFD (within 100 days post transplant) was 7%. Patients and transplant factors, including age, diagnosis, prior autologous transplant, type of conditioning regimen, type of aGVHD prophylaxis, donor type, CD34+ cell dose infused, history of diabetes or prior IFD, systemic use of steroids for GVHD and TPN administration, were examined to determine risk factors for developing IFD. In multivariable analysis, haploidentical donor transplants (HR 3.48, 95% CI 1.35–8.99, p=0.01) and the development of Grade III and IV aGVHD (HR 3.32, 95% CI 1.23–9.01, p=0.02) were risk factors for the development of IFD. Conversely, higher CD34+ cell dose infused was associated with a lower risk of IFD (HR 0.80, 95% CI 0.68–0.94, p=0.01, per 1×10⋀6 cells/kg increase). The overall mortality (OM) of patients with IFD was 41%. A higher non-relapse mortality (NRM) was observed for patients with IFD as compared to patients without IFD, 55% vs 18% (HR 3.76, 95% CI 2.03–6.97, p<0.001). In multivariable analysis, accounting for CD34+ cell count, donor type and Grade III-IV aGVHD, the effect of IFI on NRM remained significant (HR 3.32, 95% CI 1.65–6.67, p=0.001). Patients with IFD had lower overall survival (OS), 16 months as opposed to 50 months for patients without IFD. In summary, 1) The risk of IFD after allogeneic HCT was 15%; 2) Patients with IFD have a high mortality after allogeneic HCT; 3) Haploidentical donor type and development of Grades III-IV GVHD are risk factors for IFD; 4) A higher CD34+ cell dose may be protective against IFD; 5) Prior IFD did not predict for higher risk of IFD or higher mortality, suggesting these patients should not be excluded from allogeneic HCT. Further studies looking at alternate aGVHD prophylaxis regimens, strategies to increase cell dose infused and better antifungal prophylaxis for these high risk patients are needed to lessen this potentially life threatening complication. Disclosures:Mylonakis:T2 Biosystems: Research Funding; Astellas inc : Research Funding.