The prevailing philosophy in biological testing has been to focus on simple tests with easy to interpret information such as ELISA or lateral flow assays. At the same time, there has been a decades long understanding in device physics and nanotechnology that electrical approaches have the potential to drastically improve the quality, speed, and cost of biological testing provided that computational resources are available to analyze the resulting complex data. This concept can be conceived of as “the internet of biology” in the same way miniaturized electronic sensors have enabled “the internet of things.” It is well established in the nanotechnology literature that techniques such as field effect biosensing are capable of rapid and flexible biological testing. Until now, access to this new technology has been limited to academic researchers focused on bioelectronic devices and their collaborators. Here we show that this capability is retained in an industrially manufactured device, opening access to this technology generally. Access to this type of production opens the door for rapid deployment of nanoelectronic sensors outside the research space. The low power and resource usage of these biosensors enables biotech engineers to gain immediate control over precise biological and environmental data. Traditional healthcare companies market point-of-care tools with limited test libraries2. In each case, complex, analyte-specific reagents and intricate protocols create a need for multiple platforms and deep biochemical or clinical expertise to replicate the capability of a central lab3.The world is entering an inflection point in medical and biological testing with the simultaneous emergence of improved testing technology, advanced software tools, and increased expectations for quality healthcare worldwide. Organizations like the Qualcomm Tricorder XPRIZE and Gates Foundation have pushed for integrations of varied technologies in clinical tests to demonstrate potential application4. Label-free measurement tools based on field-effect sensors should remove the need for most liquid reagents, decrease power requirements, and shrink the size of handheld testing devices5. These tools will be capable of performing a wide variety of chemical and biochemical assays built on top of a single sensor manufacturing chain, leading to lower overall cost for biological measurements.There is a need for information-dense single assays that break the mold of expensive labs running colorimetric and PCR based assays6. These sensors can be described as a biologically specialized Ion Sensitive Field Effect Transistor (ISFET)7. We describe here the sensing mechanism, demonstrate a label-free capture assay, and summarize the critical manufacturing and quality control milestones met during recent sensor production.To demonstrate and validate this approach, we have commercially produced and sold a digital biosensor based on graphene-enabled Field Effect Biosensing (FEB)8. Materials like silicon require oxide layers between the transistor channel and the environment, limiting the sensitivity of field effect sensors made using those conventional materials5. Materials, such as graphene, carbon nanotubes, and molybdenum disulfide have the unique combination of chemical stability and electric field sensitivity desirable to create sensitive electronic interfaces to biological molecules9. This has led to a dense literature covering chemical and biological sensors using these materials22. Several attempts were made to produce carbon nanotube biosensors for biomedical use in the early part of the 21st century with frustrating results due to manufacturing difficulties16. Fabrication techniques using molybdenum disulfide have not matured sufficiently for devices to move beyond the proof of concept stage17.The unique attributes that are required to build effective field effect biosensors are a combination of semiconductor behavior with chemical stability of the sensor surface in air and salt water6. Here, we show devices that are manufactured in commercial fabrication facilities under industrial quality control processes. During development of our sensor design and manufacturing process, we published several papers detailing the basic stability and performance of the sensors23. In particular we have previously shown sensitivity to infectious disease biomarkers at 18 ng/mL in buffer and 500 ng/mL in serum, with sufficient specificity to be insensitive to common interfering biomarkers24. Additionally, the applicability of these sensors outside the nanotechnology community has been demonstrated via publications by independent drug discovery and biotechnology groups27. With this publication, we demonstrate for the first time, the maturation of nanoelectronic digital biosensors through a more complete description of the sensor function coupled with demonstration of scaled manufacturing and quality control processes in a commercial environment.Previously, we have shown that graphene biosensors can be reliably manufactured in research-oriented clean roomsA diagram of our device architecture is shown in Fig. g) graph in Supplemental Fig. The graphene sensor function relies on several overlapping effects, with a basic cartoon of the relevant layers shown in Fig. cs the ionic strength of the solution. The Donnan effect region extends beyond the Debye length, extending through the thickness of an ion-permeable membrane immobilized to the surface. The voltage in the bulk liquid is controlled by conventional electrochemical means. From an electrical perspective, the system can be understood with the bulk liquid as the gate of a transistor, and the combined Donnan region and Debye length as the dielectric between the graphene channel and the gate. From a biological perspective, the system can be understood as a voltage sensitive membrane incorporating proteins with driven voltages, like action potentials, in the bulk liquid. The model below explains how sensing is accomplished for binding interactions, even when charge transfer is not involved.Closest to the graphene channel, the salts in solution will form a well-organized and well-understood layer on top of the surface countering any difference in charge between the surface and the liquid. The thickness of this layer is the Debye length and is simplified in aqueous solutions to 32. This model generally relates the current (I) to typical electrical properties such as the charge carrier mobility (μ), capacitance per unit area to the gate (Cg), width (W) and length (L) of the graphene channel, source-drain voltage applied directly to the graphene (Vsd) and the gate voltage (Vg) relative to the Dirac voltage (V0). The Dirac voltage here is the gate voltage at which there is a minimum in conduction at neutral pH. The capacitance between the graene and the liquid, Cg, is a series combination of the graphene quantum capacitance, the double layer capacitance, and a capacitance across the immobilized layer due to the Donnan effect34. It is important to note that Eq. Equation The remaining terms are corrections to the gate voltage due to the influence of pH changes and the Donnan potential. This model assumes operation of the sensor near room temperature, for an equivalent gate voltage less than the Dirac voltage, for source-drain voltages below 1 V, and for a channel length greater than 10 μm.35. The surface pH sensitivity factor (α), is a material dependent value. For clean isolated graphene, α is a very low 0.02, but in practice this value is increased by the presence of oxides and nitrides used in device fabrication; α values for practical graphene transistors are around 0.3738. This term is combined with the thermal voltage (ϕth), about 26 mV, and pH shift from a neutral surface (ΔpH) to produce the equivalent gate voltage due to pH. Donnan potential (ΔφD) is created when an ion-permeable layer separates 2 collections of ions, as shown in Eq. Electronic sensitivity to pH is typically attributed to hydrogen binding to a gate dielectric. For graphene transistors, there is no gate dielectric, but this form of pH sensitivity has been shown to apply through direct shifts in the apparent Dirac voltagecx) will require an extra accumulation of a counter-ion within the layer to maintain charge neutrality. This difference in the concentration of ions between the bulk solution (cs) and that in the immobilized protein layer creates a Donnan potential39. This additional potential enables sensing beyond the Debye screening length,40 and has been demonstrated repeatedly with graphene biosensors41.In this case, the immobilized layer of proteins, peptides, surfactants, polyethylene glycol (PEG), or other soft molecules separate the graphene channel and double-layer from the bulk solution. Any charges or dipoles leading to a net charge within that immobilized ion permeable layer  and recombinant human IL-6 (IL6) were used from a commercial ELISA kit. IL-6 is a well-known cytokine and biomarker related to inflammation, autoimmune diseases and late stage cancers. Anti-IL6 was immobilized on graphene chips with a prepared carboxyl surface and activated via standard carbodiimide chemistry Fig. , as descFigure The immobilization data shows consistent trends. Addition of EDC and sNHS after calibration always leads to a decrease in current, without a change in slope. This is expected as the carbodiimide chemistry should charge the charge at the surface without adding a large amount of material, and there is no adsorbed layer to create a Donnan potential. Addition of the antibody at 30 minutes leads to a further decrease in current and a large increase in slope. This change is likely due to switching from the pH 6.0 MES buffer used during carboxyl activation to the pH 7.4 PBS pH buffer used during antibody incubation. Addition of PEG-amine after anti-IL6 immobilization always leads to a small decrease in current and a small increase in slope. This is consistent with association of PEG to the surface to serve as a blocking layer around immobilized protein. After addition of PEG, a complete layer is formed on the graphene surface, enabling a Donnan effect measurement. Addition of ethanolamine (pH 8.0) quenches any remaining activated carboxyl groups, and always causes a large decrease in current and a large increase in slope. Rinsing with PBS (pH 7.4) then raises the current and decreases the slope, although never back to the initial starting position. This repeatable set of chemistry and sensor responses indicates both reproducibility of response sensor-to-sensor as well as demonstrable, permanent change to the surface chemistry of the chips due to the immobilization process. The sign (negative or positive changes) of the responses we see during immobilization are consistent with the expected responses from the pH changes measured on bare graphene chips shown in Fig. 42. Different concentrations of IL-6 in PBS were applied to the anti-IL6 immobilized chips. The different concentrations of IL6 lead to different response magnitudes and different speeds of interaction, as expected for any kinetic binding measurement. This data compares well with the metrics provided by the ELISA kit from which the reagents were taken and previous IL-6 standard curves gathered on graphene FEB sensors43. It is notable that the data for 10 pg/mL has the expected magnitude after five minutes of measurement but has a shape which is different from the other responses. This may be the result of a pipetting error, variation in chip surface chemistry, or some protocol error. This is an example of the kind of information available in a real time assay that is not possible in an end-point assay. In addition, it highlights how process, user, and chip variation may be manifested in the data.The effectiveness of this immobilization process is demonstrated in the sensing measurements shown in Fig. When running an ELISA with this kit, the expected sensitivity is <2 pg/mL (FEB sensitivity is <2 pg/mL), and the standard range of response is between 7.8 pg/mL and 500 pg/mL (FEB range of response is 2 pg/mL to 1000 pg/mL). As shown in Supplementary Fig. The data shown here establishes that the devices we have manufactured are similar in function to other biosensors in the graphene literature. A major hurdle to providing graphene biosensors to biological researchers is the typical manufacture of nanoelectronics in research-oriented clean rooms by teams of research scientists. Previously, there was no nanoelectronic manufacturing approaching ISO9001 standards or Good Manufacturing Practices (GMP), despite those requirements for commercial and clinical applications.The facilities used to manufacture chips are located at Rogue Valley Microelectronics, Cardea, and Samtec. Rogue Valley used a 150 mm MEMS processing line, without modification to staff or equipment to process graphene wafers. The Samtec facility similarly used established silicon chip packaging equipment, staff, and traceable processes to package these graphene chips.6. The marginal cost for graphene, when using an automated system such as ours, is $0.019 per cm2. This comes from our average cost of copper foil of $0.012 per cm2 and the cost of power to run the furnaces, which in California is currently $0.007 per cm2 of grown graphene. This is less than the price of silicon wafer substrates for this work, which is $0.40 per cm2 for 150 mm wafers. The cost of processing wafers is 20-fold greater than the cost of the graphene raw material, making the processing the dominant cost factor in producing a commercial graphene device rather than the graphene raw material cost.Graphene is grown and transferred at Cardea using automation of a previously described processOur process for manufacturing digital biosensors starts with deposition and patterning of a metallization layer that creates the routing for the source-drain voltage on 15 graphene transistors per die, as well as the platinum reference and counter electrodes. Graphene is grown and transferred onto the wafer. After deposition, graphene is etched using a hard mask and an oxygen plasma. A silicon nitride encapsulation layer is deposited via plasma enhanced chemical vapor deposition (PECVD). Encapsulation here refers to deposition of a dielectric barrier layer across nearly the entire chip that is intended to prevent mechanical and chemical damage during the chip packaging and printed circuit board (PCB) assembly processes. Windows are etched into the silicon nitride down to the graphene hard mask. Wafers are diced and packaged via a chip-on-board process, where an epoxy is used to protect wirebonds and define the liquid wetted area. After packaging, the chips are annealed and undergo strict quality assurance (QA) processes to ensure reproducible quality.Figure Differential interference contrast microscopy is used along with AI-enabled defect identification to perform optical evaluations, such as those shown in Fig. By far, the largest source of defects is polymer contamination resulting from the graphene transfer process. However, the largest source of defects that effect the operation of the chip are major tears, which may come from a variety of sources including handling, the graphene transfer process, or dust. Lithography defects are relatively rare, and primarily along the wafer edge. Other types of defects include process errors not associated with lithography, graphene double layers, or unusual forms of contamination.Vsd. This low power draw anticipates the need for thousands of chemically differentiated sensor channels or other graphene transistors in a portable form factor.In addition to focusing on use of QA tools that are current standards in commercial fabrication facilities, we also focus on optimizing the stage at which QA measurements are performed. Figure 44.At the end of the QA process, a map of yielded dies on the wafer is produced. Figure 45.We present the first complete characterization of this fusion of electronics and biochemistry. This marks a break from the past few decades of great feats of artisanal nanoelectronics and demonstrates the potential for graphene biosensors to serve as the interface between biochemistry and digital analysisA core challenge has been making the transition from an expensive, variable sensor to a cost-efficient, consistent batch-to-batch manufactured sensor. A key to that transition is following a rigorous specification and QA process appropriate for a biologist end user rather than a nanotechnologist. While the manufacturing and QA processes described here were used to create graphene-based biosensors, these processes are broadly enabling for many electronics applications using graphene and other nanomaterials.The demonstrated biological sensing capability, low power requirements, and compact size of graphene-based biosensors will enable development of the next generation of biochemical applications. With the most difficult piece of the puzzle – cost-effective large-scale manufacturing – solved, low-power, portable digital biosensors can significantly impact healthcare industries with innovative new products that enable cutting-edge life science research, drug discovery applications, and diagnostic and health monitoring platforms.Vsd was held at 10 mV. An example of the raw data measured this way is shown in Supplemental Fig. The Agile R100 system from Nanomed (a Cardea owned brand) was used for all measurements. The standard electrical settings were used. The gate voltage was swept between ±100 mV in a triangle wave at a slow speed of 0.3 Hz, while 47. For ionic strength measurements, deionized water was used for calibration, and concentrations of NaCl from 0.25 M to 2 M in deionized water were used for the measurement. Agile FLEX biosensor chips (Nanomed) were used for pH and ionic strength measurements.Except where noted, 1X phosphate buffered saline solution pH 7.4 (PBS) (ThermoFisher # 10010031) was used for calibration and measurement. Hydrochloric acid (HCl) was used to create PBS solutions of varying pH from 5.8 to 7.4, measured with a calibrated glass electrode pH meter. Fifty millimolar 2-(N-morpholino)ethanesulfonic acid buffer pH 6.0 (MES)  was used for crosslinking chemistry. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (Amresco # N195) was used with sNHS (N-hydroxysulfosuccinimide) (G Biosciences # BC97) to activate carboxyl groups for amine attachmentAnti-IL6 and IL6 were purchased as part of an ELISA kit (ThermoFisher # 88-7066-88).Agile COOH biosensor chips (Nanomed) were used for IL6 measurements. COOH chips were prepared via incubation of clean graphene chips with 3 mM pyrene-carboxylic acid (TCI # P1687) in methanol for two hours.41. Finally, the biosensor chips were rinsed 5 times in PBS, and the last rinse was incubated for 5 minutes to stabilize the signal prior to measurement. This process was performed with a hand pipette following software prompts. Additional testing with DMEM/FBS was performed and is shown in Supplemental Fig. The detailed process for anti-IL6 immobilization is: After calibration in 50 mM MES pH 6.0 for 5 minutes, 2.08 mM EDC and 5.53 mM sNHS in 50 mM MES pH 6.0 was incubated for 20 minutes. The chips were rinsed 2 times with 50 mM MES pH 6.0, followed by 14.6 nM anti-IL-6 in 1X PBS pH 7.4 for 15 minutes. Then, 3 mM PEG-amine (Broadpharm Item #BP-22355) in PBS was incubated for 15 minutes to block the surface. A blocking layer such as PEG is necessary to control non-specific surface chemistry. The chips were then incubated with 1 M ethanolamine pH 8.0 (Alfa Aesar Cat# L14322) for 15 minutes to quench any remaining carboxyl groups. A quench step is used in standard carbodiimide chemistry to remove activated carboxyl sites that may link to amines present in the test samplePrime Si, 150 mm, P-type wafers were purchased from Rogue Valley Microdevices, as was all mask fabrication and lithography. A wet thermal oxide of 3,000 Å was first grown. Metallization layers of 100 Å Cr and 500 Å Pt were patterned via liftoff technique.Graphene was grown via chemical vapor deposition at Cardea on copper and transferred to the wafer via bubble transfer. Microscopy was used to evaluate graphene quality at every transistor location prior to further processing.At Rogue Valley Microdevices, a 1,000 Å Au layer was deposited on the graphene and etched to form a hard mask. An oxygen plasma etch was used to remove excess graphene. A 5,000 Å SiN layer was deposited via PECVD, patterned and etched via RIE.Custom PCBs for packaging were designed by Varasco Engineering, laid out by Pacific Design, and purchased from Consisys. Samtec diced wafers after microfabrication and performed a standard chip-on-board packaging process using gold wirebonds and a custom dam-and-fill encapsulation pattern to create the liquid well on the sensor.The chips were cleaned, vacuum annealed at 200 °C, and inspected at Cardea.Optical microscopy was performed using an nSPEC microscope (Nanotronics). AI driven automated defect identification software was also provided by Nanotronics.Electrical QA was performed using an Agile R100 (Nanomed) in QA mode.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


The existence of a sarcoidosis-lymphoma syndrome has been previously proposed since the relation between sarcoidosis and an increased risk of lymphoproliferative disorders is well established. Multiple myeloma is a malignant multifocal proliferation of clonal plasma cells within the bone marrow, and its association with sarcoidosis has been rarely described. We present a concurrent diagnosis of sarcoidosis and multiple myeloma and make a brief analysis of the reported cases in the literature. A 65-year-old woman underwent surgery for the excision of a wrist mass that presented 3 years before. Histological analysis showed sarcoid-type epithelioid granulomas without necrosis, establishing soft tissue sarcoidosis. Further evaluation revealed marked interstitial lung parenchyma lesions and large intrathoracic adenopathies. Bronchofibroscopy with transbronchial biopsy confirmed lung sarcoidosis. In addition, blood analysis showed monoclonal IgG kappa gammopathy. A bone marrow biopsy confirmed hypercellularity with 60% plasma cells and plasmocyte infiltration. Thus, the diagnosis of systemic sarcoidosis and multiple myeloma was established simultaneously. In a brief review of the literature, we identified 33 reports of cases with both sarcoidosis and multiple myeloma. We point out the importance of a high level of suspicion for the association of sarcoidosis with malignant haematological diseases such as multiple myeloma. Sarcoidosis is a T-cell-mediated immunological response against an unknown environmental trigger in a susceptible host. Noncaseating granulomas, consisting of compact and centrally organized collections of CD4+ T cells, macrophages, and epithelioid cells, are the hallmark of sarcoidosis and the outcome of an unbalanced T-helper 1 immune response . MultiplA 65-year-old Caucasian women, retired cook, was referred to our outpatient clinic due to a three-year history of a painless mass on the dorsal side of the right wrist. Her medical background was positive for type 2 diabetes mellitus, dyslipidaemia, bronchitis, and venous thrombosis of the right eye. The wrist lesion had been growing gradually and affected finger movement. There was no history of blunt or penetrating trauma.erythema nodosum). There were no other palpable lymph nodes, hepatosplenomegaly, fever, night sweats, or constitutional symptoms.An ultrasound, complemented with nuclear magnetic resonance, identified a lesion on the dorsal plane of the right wrist, extending to the inner face, but without vascular or tendinous invasion, measuring about 100 × 60 × 18 mm in diameter and having well-defined limits . After s9/L (reference range 4–11 × 109/L), C-reactive protein of 0.87 mg/dL (reference range <0.30 mg/dL), erythrocyte sedimentation rate of 83 mm (reference range <20 mm), serum angiotensin converting enzyme (ACE) of 141.28 U/L (reference range 12–68 U/L), albumin of 3.66 g/dL (reference range 3.97–4.94 g/dL), and total serum protein level of 8.40 g/dL (reference range 6.40–8.20 g/dL). Serum protein electrophoresis revealed a monoclonal band, confirmed by immunofixation to be immunoglobulin IgG kappa. Further quantification of serum immunoglobulins showed an elevated IgG level of 3120 mg/dL (reference range 70–1600 mg/dL), with normal IgM and IgA, and beta-2 microglobulin of 2.09 mg/dL (reference range 0.67–1.31 mg/dL). Mantoux assay and Bence Jones proteinuria quantification were negative, and both renal function and serum calcium were within the normal range. The screening for HIV and hepatitis B and C was negative.Laboratory studies showed haemoglobin 9.7 g/dL (reference range 11.5–16.5 g/dL), white blood cells count of 4.6 × 10A high-resolution computed tomography of the chest showed large mediastinal and axillary adenopathies with extensive conglomerates, associated with marked and diffuse permeability of the entire pulmonary parenchyma, thickening of the interlobular septa, and fine bronchovascular and subpleural micronodularity .The patient's lung function testing revealed a mild restrictive pattern with decreased diffusion capacity. A bronchofibroscopy with transbronchial biopsy and bronchioalveolar lavage was performed. Respiratory tract fluid analysis showed an increased CD4/CD8 ratio of 9.02, and both acid-fast staining (Ziehl–Neelsen coloration) and mycobacterial culture were negative. Bronchial biopsy histological results revealed mild chronic inflammatory infiltrate with multiple nonnecrotizing sarcoid-type epithelioid granulomas and giant multinucleated Langerhans cells Figures . The patHence, a simultaneous diagnostic of systemic sarcoidosis and multiple myeloma stage I according to the International Staging System was established. The patient was started on prednisolone 60 mg/day with clinical and biochemical improvement. Bone marrow plasma cell infiltration dropped to 28%, and IgG immunoglobulin and ACE returned to normal values. Although a slow reduction of steroid therapy (to 5 mg/day) was achieved in time, previous oral antidiabetic therapy became insufficient and prompted insulin introduction.Only few cases of sarcoidosis in association with MM have been reported in the literature, so there is not enough scientific evidence available to support a causal relation for the occurrence of these two diseases. Nonetheless, epidemiological data highlight the higher risk for lymphoproliferative disease development in sarcoidosis patients –8.Brincker and Wilbek  publisheIn our review of the literature, 33 cases with both sarcoidosis and MM were identified although patient characteristics and diagnostic information were not available for all of them , 6–25. DH17 cells and Treg cells in sarcoidosis and MM immune mechanisms [The explanation for the relationship between sarcoidosis and lymphoproliferative disorders, particularly MM, is still controversial and unknown. One possible link between sarcoidosis and MM is the polyclonal hypergammaglobulinemia induced by sarcoidosis chronic inflammatory response. This continuous state of stimulation results in prolonged B cell and plasma cell half-life, which in addition to immune system imbalance in sarcoidosis patients, predisposes for genetic mutations and might lead to haematological malignancy development , 20. RecIn our case, sarcoidosis diagnosis was first made after a soft tissue lesion excision in a 65-year-old woman. Although both sarcoidosis and MM were diagnosed simultaneously, previous respiratory symptoms suggest that sarcoidosis pulmonary involvement was already present years before and misdiagnosed as bronchitis. This fact might also account for the older age at the time of the diagnosis of sarcoidosis. No usual abnormalities related to MM, such as lytic lesions or renal function impairment, were present at the time of diagnosis, and only the serum protein electrophoresis monoclonal band called the attention to a possible underlying disease other than sarcoidosis. We aim to call the attention to the possibility of LD development in patients with sarcoidosis. A high level of suspicion should always be present both at the time of diagnosis as well as during follow-up since LD seems to develop years after the diagnosis of sarcoidosis.


Health service change is difficult to achieve. One strategy to facilitate such change is the clinical pathway, a guide for clinicians containing a defined set of evidence-based interventions for a specific condition. However, optimal strategies for implementing clinical pathways are not well understood. Building on a strong evidence-base, the Psycho-Oncology Co-operative Research Group (PoCoG) in Australia developed an evidence and consensus-based clinical pathway for screening, assessing and managing cancer-related anxiety and depression (ADAPT CP) and web-based resources to support it - staff training, patient education, cognitive-behavioural therapy and a management system . The ADAPT Portal manages patient screening and prompts staff to follow the recommendations of the ADAPT CP. This study compares the clinical and cost effectiveness of two implementation strategies (varying in resource intensiveness), designed to encourage adherence to the ADAPT CP over a 12-month period.This cluster randomised controlled trial will recruit 12 cancer service sites, stratified by size , and randomised at site level to a standard (Core) versus supported (Enhanced) implementation strategy. After a 3-month period of site engagement, staff training and site tailoring of the ADAPT CP and Portal, each site will “Go-live”, implementing the ADAPT CP for 12 months. During the implementation phase, all eligible patients will be introduced to the ADAPT CP as routine care. Patient participants will be registered on the ADAPT Portal to complete screening for anxiety and depression. Staff will be responsible for responding to prompts to follow the ADAPT CP. The primary outcome will be adherence to the ADAPT CP. Secondary outcomes include staff attitudes to and experiences of following the ADAPT CP, using the ADAPT Portal and being exposed to ADAPT implementation strategies, collected using quantitative and qualitative methods. Data will be collected at T0 , T1 (6 months post Go-live) and T2 (12 months post Go-live).This will be the first cluster randomised trial to establish optimal levels of implementation effort and associated costs to achieve successful uptake of a clinical pathway within cancer care.ACTRN12617000411347The study was registered prospectively with the ANZCTR on 22/3/2017. Trial ID  Anxiety and depression are the third largest causes of disability in Australia, impacting on family and social functioning, work performance, suicidal ideation, and survival , 2. RateBecause anxiety and depression are often under-detected, routine screening of all cancer patients for psychological distress using validated, reliable, objective measures, is internationally endorsed . The IntScreening and detection, however, are only part of the solution. Several recent systematic reviews –12 highlA clinical pathway for screening, assessment and management of anxiety and depression in adult cancer patients (ADAPT CP) was developed to guide best practice in Australia . The ADAThe ADAPT CP recommends formalised routine screening for anxiety and depression using the Edmonton Symptom Assessment System (ESAS-R)  or DistrWhile clinical pathways have shown considerable success in bringing about change in patient management, the context and implementation strategies utilised when introducing them into routine care are critical to success , 20. FaiIn response to these identified barriers, our team developed and tested a number of online resources to support implementation of the ADAPT CP, including a web-based management system to operationalise the pathway tailored to site-specific staffing and resources , patient education materials, health professional education modules and an online cognitive-behavioural therapy program for anxiety and depression in patients with cancer (iCanADAPT) .The ADAPT Portal prompts patients to complete validated screening and assessment questionnaires; alerts health professionals by system flags and email when a patient has elevated anxiety or depression; describes the pathway relevant to that individual patient and appropriate staff responses; provides standardised templates for referral and progress review to/from mental health services when required; and schedules ongoing follow up screening of patients at pivotal times/visits. The ADAPT Portal was pilot-tested in early 2017 (paper under review) and shown to be feasible, technically operational and acceptable.The ADAPT patient education materials explain the importance of screening for anxiety and depression, the process involved for patients in the use of the ADAPT CP and Portal, and links to existing patient/caregiver resources about cancer-related anxiety and depression. A pilot study using qualitative methods with 20 current cancer patients and survivors showed the materials to be acceptable, and at an appropriate reading level (paper under review).The ADAPT online health professional education modules were developed using evidence-based learning principles and spaced education to promote interactive learning. This education targets screening, assessing severity, making a referral - particularly when patients are reluctant - and empathic communication. Modules are available on eviQ Cancer Education Online (www.eviq.org.au), a widely used Oncology education site in Australia, via links in the ADAPT Portal. These education modules were pilot tested in a pre-post simulation study with 12 Oncology nurses and found to be acceptable, feasible and to associated with increased skills (paper in preparation).A program delivering online cognitive-behavioural therapy for anxiety and depression has been developed for patients with early stage cancer (iCanADAPT Early). The therapy includes behavioural activation, cognitive restructuring and problem solving, and mindfulness strategies, completed in 6–8 sessions over 10–12 weeks. This online program is available in the ADAPT Portal for patient self-referral or clinician referral. Participants complete a short screening instrument (the K-10) [he K-10)  before ehe K-10)  and evalThere is a growing evidence-base that various strategies  increase the likelihood of successful implementation . HoweverIn this study we aim to determine the optimal “dose” of implementation effort and cost required to effectively achieve adherence to the ADAPT CP. Thus, cancer services participating in the study will be randomised to a standard (Core) implementation strategy versus a supported, tailored (Enhanced) implementation strategy for the ADAPT CP.The primary objective of the ADAPT Cluster randomised controlled trial (Cluster RCT) is to evaluate the efficacy and cost-effectiveness of two implementation strategies (Core versus Enhanced) in achieving adherence to the ADAPT CP.i.describe adherence to each component of the ADAPT CP and predictors of adherenceii.describe the acceptability, adoption, appropriateness, feasibility and fidelity of individual implementation strategies and resources used to support implementation of the ADAPT CP in cancer services in NSWiii.evaluate staff and patient acceptability of the ADAPT CP, within a NSW health contextiv.describe the costs of the ADAPT CP and compare the costs of the two implementation strategies (Core vs. Enhanced) used to facilitate uptake of the ADAPT CPv.evaluate the cost effectiveness of two implementation strategies (Core vs. Enhanced) used to facilitate uptake of the ADAPT CPvi.describe levels of anxiety and depression of patients participating in the ADAPT CP over a 12-month period, and patients’ allocation to five clinical, subclinical and non-clinical categories of anxiety and depression by staffvii.describe referral patterns of cancer services before and after the introduction of the ADAPT CP, including uptake of referrals by patients.Secondary objectives are to: will be greater in the Enhanced implementation arm than in the Core implementation arm.Adherence to each component of the ADAPT CP (yes/no) will be greater in the Enhanced implementation arm than in the Core implementation arm.Adherence to the ADAPT CP will increase from the 6- to 12-month follow-up.Staff-perceived individual and site readiness to change will be associated with adherence to the ADAPT CP.Staff use of the ADAPT resources will be associated with adherence to the ADAPT CP.Characteristics of the service , will influence staff attitudes and adherence to the ADAPT CP.Greater awareness/use and perceived utility of implementation strategies will be associated with better adherence to the ADAPT CP.Adherence to the ADAPT CP and predictors of adherence7.The ADAPT CP will be acceptable to staff and patients.8.At least 60% of staff will be aware of, use, and rate as useful, the ADAPT resources .Acceptability of the ADAPT CP and resources9.Both Core and Enhanced ADAPT implementation strategies will be feasible (delivered) and delivered with fidelity as planned.10.At least 60% of staff will be aware of, use, and rate as useful, both Core and Enhanced ADAPT implementation strategies.11.Acceptability, adoption, appropriateness, feasibility, fidelity, and penetration of implementation strategies will be greater in the Enhanced implementation arm than the Core implementation arm.12.Improvements in staff-reported acceptability of the ADAPT CP and perceived readiness to change will be greater at 6- and 12-month follow-ups in the Enhanced implementation arm compared to the Core implementation arm.ADAPT implementation strategies (Core and Enhanced)13.While more costly, the Enhanced implementation arm will achieve greater adherence than the Core implementation arm.Cost-effectiveness14.Anxiety and depression screening scores at rescreening will be significantly lower than at the initial screen.Patient outcomesThe study is a Cluster RCT being undertaken in the state of New South Wales (NSW), Australia. NSW health services are managed through 15 local health districts and three specialty networks. Twelve cancer services will be stratified by size of service - large (≥100 new pts./year) versus small (< 100 new pts./year)) to ensure equal patient volume in the two study arms. The sites will then be randomised at the cancer service level to a Core implementation strategy arm versus an Enhanced implementation strategy arm to facilitate uptake of the ADAPT CP.After a 3-month period of site engagement, staff training and tailoring of the ADAPT CP and ADAPT Portal for each participating cancer service (site), that site will “go-live” and implement the ADAPT CP for 12 months, as part of routine care. ADAPT research staff will deliver the Enhanced or Core implementation strategies over that period, according to randomisation. The study uses a mixed methods design. Both quantitative and qualitative data eliciting staff attitudes to and experiences of the ADAPT CP and implementation strategies utilised, will be collected at T0 , T1 (6 months post go-live) and T2 (12 months post go-live).Taking into account the governance, engagement and implementation phases, each site will participate in the study for a period of 18 months. Three sites will be activated every 3 months. Thus, all sites will have completed the study over a 30 month/2.5-year period.Cancer services will be eligible to participate if they provide care for at least 100 patients per year in NSW Australia. Eligible cancer services may operate within the public or private healthcare system. They may be whole services or single departments within services, such as tumour streams (e.g. breast cancer or haematology) or treatment streams . If there is more than one department/hospital which shares common staff members, they will be considered one site for the purpose of this study. This is necessary to ensure that participating staff members are not exposed to both Core and Enhanced implementation strategies.endorsing and enabling staff training for ADAPTengaging with ADAPT staff for the 3-month engagement and tailoring periodallowing a complete site profile audit and release of their organisational chartauthorising release  of Medical and Pharmaceutical Benefit (MBS/PBS) provider information and statistics;and if they do not have Wi-Fi/broadband/internet access and/or an internet browser version that complies with the ADAPT Portal requirements.Cancer services will be excluded if they are unable to commit to:An initial meeting (face to face or via teleconference) will be held, attended by representatives of the ADAPT research team, the Head of the cancer service, and representatives of the psychosocial, nursing, clinical trial and IT staff. At this meeting, the study will be described in detail, and potential interest in participation will be ascertained. Interested cancer services will be asked to identify a local ADAPT champion, and to sign an ADAPT Research Participation Agreement.All staff employed on a permanent or ongoing (6 months or more) basis, either full or part time or as a visiting medical officer, in a role that provides clinical care to patients with cancer or client focussed administrative support or IT/managerial support, will be invited to participate in the study. Staff will be identified as part of the organisation chart mapping exercise during site engagement. Site champions will identify and approach eligible staff, to whom they will explain the study. Interested staff will be contacted by the research team and provided with a written information sheet and consent form to sign.A site, or individual cancer service staff member, may withdraw from the study and elect to have their data removed from the study database. If a site chooses to withdraw prior to completion of baseline data collection, and there is sufficient time within the study timeline, the site will be replaced by a reserve list site pre-randomised to the same study arm.All patients at participating sites receiving psychosocial care related to their cancer diagnosis will be treated according to the ADAPT CP as part of routine care during the 12-month implementation period. If a patient does not wish to complete screening on a particular occasion (defer) or declines screening completely, this will be recorded in the ADAPT Portal and noted within the patient’s medical record, in line with usual record keeping practices at participating cancer services.Patients participating in the ADAPT CP will be asked to give informed consent for the researchers to access their medical records and/or Medicare and Pharmaceutical Benefits Scheme (MBS/PBS) claims history for a period of 10 years to enable health economic analyses. Patients may withdraw this consent at any time.ADAPT PortalADAPT Health professional trainingADAPT Patient education materialsiCanADAPT Cognitive-behavioural TherapySites will be randomised to a Core implementation arm versus an Enhanced implementation arm to facilitate uptake of the ADAPT CP. All cancer services will receive access to, and training to support use of, the online resources developed to support ADAPT. These resources (described in detail above) are the:Core: These sites will receive implementation strategies consistent with usual roll out of a clinical pathway in the NSW health context, to inform staff of the ADAPT CP and enable them to initiate use of the ADAPT Portal and intervention resources. Once the ADAPT Portal is launched and the implementation phase has commenced, Core sites will receive reporting feedback and audit reports at 4 weekly intervals and additional support from the ADAPT team only at their request (a passive approach).Enhanced: These sites will receive the same implementation strategies as the core group, with the addition of more prolonged, active engagement of the ADAPT team, that will continue during the 12-month implementation phase. These additional strategies will include monthly meetings to identify training and support needs of local champions, additional awareness activities, audit and feedback reports prior to and midway through the implementation phase summarised and presented face-to-face by research staff to site staff, and other tailored strategies to address the specific barriers and facilitators in each site (an active approach).The Core implementation and Enhanced implementation arms will differ in the implementation strategies used to support the use of ADAPT CP at their cancer service. Specifically, these differences are:The strategies, described in detail in Table Randomisation will be performed by the study statistician using online randomisation software (STATA). Allocation concealment will be preserved; both sites and ADAPT Program staff conducting assessments will remain blinded until study completion. Following randomisation, sites will undertake a 3-month period of engagement to clarify study procedures, train staff in the ADAPT CP and use of the ADAPT resources, and tailor the ADAPT Portal to local site resources and preferences. Study sites will then complete baseline (T0) staff questionnaires and interviews to ascertain staff views on service and staff readiness to adopt the ADAPT CP. The online questionnaires will be available on RedCap (Research Electronic Data Capture)  and autoSites will then “go-live,” integrating the ADAPT CP into clinical care for a 12-month implementation period. Staff will be encouraged to introduce the ADAPT CP to all patients as part of routine care, and on their verbal consent, register them into the ADAPT Portal. Patients will then be asked to access the ADAPT Portal for efficient screening and data collection, information and access to ADAPT resources. Staff will be asked to respond to ADAPT emails and alerts for patients who require further assessment and referral. ADAPT research staff will administer Core versus Enhanced implementation strategies as per randomisation during this period.Mid-way through the 12-month implementation period (T1 at 6 months) and at the end of the implementation period (T2 at 12 months), questionnaire data will again be collected from cancer service staff, and a subset of cancer service staff will participate in an audio-recorded semi-structured interview to ascertain staff views on implementation outcomes, and awareness of and perceived utility of the ADAPT CP implementation strategies.Timing of enrolment, engagement, intervention and data collection is shown in Table Case report forms (CRFs) in this study will take the form of site profile forms, contact logs, implementation strategy checklist and observational diaries, implementation strategy data psycho-social activity data and Medicare Data forms completed by ADAPT Program staff to collect service level data and staff surveys completed by cancer service staff participants. All other quantitative data will be collected electronically via the ADAPT portal. Additionally, the research team will conduct internal audits by reviewing automated reports built into the ADAPT Portal to ensure system functioning, correctness and accuracy of data entry.The primary outcome is site adherence to the ADAPT CP, operationalised as the percentage of patients at each site whose care is considered adherent to the ADAPT CP, using data extracted from the ADAPT Portal. The measure, guided by the literature on adherence , captureADAPT CP secondary outcomes will address the following domains: acceptability, adoption, appropriateness, feasibility, fidelity, cost and penetration, and sustainability of the ADAPT CP, defined in large part by the Proctor framework of implementation outcomes  see det. ADAPT iAcceptability is operationalised as the perception of cancer service staff that the ADAPT CP (1) and the ADAPT implementation strategies (2) are agreeable, palatable and satisfactory, the first in addressing anxiety and depression in their client population, the second in facilitating implementation. This will be measured qualitatively during staff interviews.Adoption of the ADAPT CP (1) is operationalised as the proportion of patients at each site who are registered and screened on the ADAPT Portal. Adoption of the ADAPT implementation strategies (2), is operationalised as mean staff ratings of how much they were aware of, observed and used each implementation strategy, collected via the staff questionnaire.Appropriateness is operationalised as the extent to which staff believe that the ADAPT CP has fit, relevance and compatibility at the level of their setting, their role, and the needs of their consumers, measured quantitatively by the Hands 4 U questionnaire [ionnaire   will be calculated from the research team logs. Costs of developing, maintaining and delivering different components of the ADAPT CP, will also be calculated. Patient health services use, including pharmaceutical prescriptions related to anxiety and depression, will be extracted from Medicare MBS/PBS and hospital medical records data. These data will be used to estimate changes in health system resource use before and after participating in the ADAPT CP, and to determine whether differences in adherence are associated with differences in health system resource use and overall costs.Penetration is operationalised as the number of staff and services who were aware of, and engaged with, the ADAPT CP and implementation strategies.Sustainability, defined as the extent to which the ADAPT CP is maintained or institutionalized within a service setting’s ongoing, stable operations, will not be measured during the ADAPT Cluster RCT, as no data will be collected beyond the 12 months implementation phase. However, intention to sustain the ADAPT CP will be elicited during T2 qualitative interviews. Further funding will be sought to audit actual sustainability in the future at participating sites, post the RCT.Predictors of adherence, including staff attitudes to the ADAPT CP, perceived social influence to enact it, self-efficacy, organisational readiness and intention to implement the ADAPT CP, will be assessed by the Organisational Readiness to Change (ORIC) questionnaire [ionnaire , Hands4Uionnaire  and someDemographic and disease factors will be collected including years in oncology practice and years in current position (staff only); age, gender, marital status, education, postcode, ATSI status (staff and patients); and cancer type and stage of disease (patients only).Additional secondary outcomes include the prevalence of anxiety and depression over time, accessed from the ADAPT Portal.All adverse events (AEs) and serious adverse events (SAEs) related to implementing the ADAPT CP will be recorded concisely using standard terms.The interventions being evaluated in this study are targeted at cancer service staff. It is not envisaged that cancer service staff will experience any adverse events. In the unlikely occurrence that attendance at a training or information session, completion of a survey or an interview does result in an adverse event, the relevant ethics committee will be notified. As screening and management of anxiety and depression will be introduced as standard practice, the reporting of adverse events related to anxiety and depression will not be reported as a study related AE event. There are no safety endpoints and no criteria for early study termination.All data will be stored in locked filing cabinets in a locked office at The University of Sydney or scanned and stored in password protected files and kept at the University of Sydney on a secure server. Electronic data will be password protected and kept at The University of Sydney on a secure server, which is backed up daily. Study-related records for all participants will be retained in a secure storage facility for at least 7 years after the completion of the research, according to the National Health and Medical Research Council requirements. These include: participant files, study protocol, signed consent forms, CRFs, questionnaires, ethics correspondence and approvals, other regulatory documentation, and other documents pertaining to the conduct of the study. All relevant documentation according to the Data Management Plan will be held by the lead investigator at each participating site for the duration of the study. This study will adhere to PoCoG’s quality assurance processes, including adherence to standard operating procedures and regularly reporting of study progress to the organisation.https://www.cancerinstitute.org.au/data-research/data-held-by-cinsw/nsw-cancer-registry), approximately 37,525 patients in NSW will be diagnosed with cancer in NSW each year. Based on prior epidemiological studies, 42–45% and 35–37% of these will experience mild depression or mild anxiety, respectively [Based on cancer registry data for NSW (2009) , there is at least 80% power to detect a difference in adherence rates of 30% in the Core and 60% in the Enhanced arm using a two-tailed test with Type 1 error rate of 5%. The sample size has been adjusted for the variance inflation factor due to the clustered design and can accommodate an intraclass correlation coefficient (ICC) as high as 0.14 to achieve 80% power and 0.10 to achieve 90% power , 39. AltSecondary outcomes will use data from either the ADAPT Portal, relating to patient activity through the ADAPT CP, MBS/PBS, staff surveys, staff interviews, or ADAPT research team observational records. Given the expected large sample size of 5568 patients, power should be adequate to test the patient activity related secondary outcomes. For secondary outcomes using staff reported data we anticipate a minimum of 10 staff per site, with 12 sites participating (120) in total.The primary analysis will be conducted using a generalized estimating equations (GEE) approach to account for the correlation of outcomes within a cluster (site) using an exchangeable covariance structure and a bias correction factor to account for the small number of clusters . The depSecondary outcomes for this study include the collection of qualitative and quantitative data. Qualitative data will be analysed using content and thematic analysis. This will apply to data collected from (i) cancer service staff interviews and (ii) observational diary and contact log data captured by the ADAPT research team. Qualitative data will be coded by researchers trained in qualitative methods. Initial themes and subthemes will be identified and discussed by members of the research team to establish a coding tree. Transcripts and/or diaries and/or contact logs will then be coded by a minimum of two coders. Coding discrepancies will be resolved by discussion.Quantitative data will be analysed using descriptive statistics, univariate and multivariate analyses. Analyses will be conducted at patient level and site level. Generalized estimating equations (GEE) will also be used.International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Good Clinical Practice (ICH-GCP),The National Health and Medical Research Council National Statement on Ethical Conduct in Human Research,The Australian Code for the Responsible Conduct of Research,Therapeutic Goods Administration’s Australian Clinical Trial Handbook, andDeclaration of Helsinki.This study will be conducted in compliance with the:This study will advance the field by identifying the level of implementation support, and resource use (Core versus Enhanced) required to effectively achieve adherence to a clinical pathway for anxiety and depression in cancer patients (ADAPT CP). It will be the first study internationally to explore this research question through a rigorous cluster randomised controlled trial, where sites are randomised to different implementation strategies. The costs and consequences of the implementation strategies will also be evaluated. The study will describe predictors of adherence, including staff and service readiness for change, staff attitudes, self-efficacy, social influence and intention to implement the CP, and staff, patient and service characteristics. A strength of the study is that adherence data, as well as potential predictors and cost data, will be collected longitudinally, allowing these factors to be explored over time.The ADAPT CP is relevant to all cancer types and cancers of any stage. The ADAPT Cluster RCT trial will recruit cancer services providing care to a range of tumour streams, and it is intended that the ADAPT CP will be applied to all patients within these services, including those treated with curative intent and those treated palliatively. Furthermore, randomisation will be stratified by size of service to ensure inclusion of both large and small cancer services, and those within both metropolitan and rural/regional areas, to determine the implementation strategies required across a range of services likely to vary in their resources and culture. While this study is restricted to the ADAPT CP, the findings will be generalisable to other CPs, providing a broader impact on health service change.A qualitative component is included in this study to capture the complexity of the barriers and facilitators influencing uptake of the ADAPT CP, and response to the ADAPT implementation strategies. These are expected to vary across sites with different clinical and research cultures, resources, patient populations and staffing profiles, and qualitative research has value in understanding these issues.The two levels of implementation effort tested here (Core implementation strategy versus Enhanced implementation strategy) include multiple types of implementation strategies, and it will not be possible to determine whether one versus another has had the greatest impact. However, we will be able to investigate potential mediators and moderators of intervention success, to guide hypotheses for investigation in future studies.With an increased focus on implementation of evidence that has been costly to accrue, and the potential to improve patient outcomes immediately, clinical pathways are likely to form the basis for care in a growing number of health systems. Clinical Pathways have great potential for enhancing the translation of evidence into practice, however health system policy and planning require accurate data on the costs and size of implementation effort required to ensure their success. Armed with such data, health service administrators can better plan introduction of clinical pathways within current budget constraints. Thus, this study will provide information critical to improving healthcare outcomes.


Staphylococcus aureus clonal complex 398. Genes encoding classic virulence factors were absent. The patient made a slow recovery after multiple surgical interventions and a protracted course of intravenous flucloxacillin.We report a case of severe disseminated infection in an immunocompetent man caused by an emerging lineage of methicillin-sensitive  Staphylococcus aureus (MRSA) clonal complex 398 (CC398) lineage, associated with livestock, has been a global public health concern (Staphylococcus aureus (MSSA) CC398. Evidence suggests MRSA CC398 and MSSA CC398 are of distinct lineages , platelets 113 ×109/L, C-reactive protein 437 mg/dL, creatine kinase 1,277 IU/L, lactate 4.5 mmol/L, and albumin 19 g/L. Laboratorians isolated MSSA from blood cultures taken at admission. Antimicrobial susceptibility testing using disc diffusion methodology  showed susceptibility to cefoxitin, rifampin, ciprofloxacin, trimethoprim, and tetracycline, but resistance to penicillin, erythromycin, and clindamycin. Oxacillin MIC testing confirmed flucloxacillin sensitivity (MIC 0.38 mg/L).Shortly after arriving in the United Kingdom, a 60-year-old man from Colombia sought medical care after experiencing malaise, sore throat, and joint and muscular pain for 7 days. At hospital admission, he had signs consistent with sepsis and evidence of spreading soft tissue infection. Results of admission blood tests included leukocytes 49.9 × 10Computed tomography and magnetic resonance imaging (MRI) images taken at admission indicated pyomyositis of the left subscapularis, inferior scalene, and intercostal muscles; a large retropharyngeal (The patient was treated with intravenous flucloxacillin (2 g every 4 h) and rifampin (300 mg 2×/d) throughout his hospital admission. Initial surgical interventions consisted of incision and drainage of the retropharyngeal, right deltoid, and left subscapular collections. A second MRI, taken on day 13 of admission, showed discitis and osteomyelitis of the C3 and C4 vertebrae, return of the retropharyngeal collection, and enlargement of the psoas and epidural collections, with evidence of compression of the distal cord. No neurosurgical intervention was undertaken because the patient showed no focal neurologic signs. Despite images showing initial enlargement of collections, the patient experienced a slow clinical and biochemical recovery. MSSA was recovered from blood cultures until day 10, but tests were negative thereafter. A second computed tomography image, taken on day 30, showed a reduction in the size of the epidural and psoas collections. The patient returned to Colombia on medical grounds on day 46 to continue intravenous antibiotic therapy. tst), exfoliatins, or Panton-Valentine leukocidin. Presence of the immune evasion cluster genes and the canonical single-nucleotide polymorphisms described by Stegger et al. (blaZ and erm(T) were identified, correlating with the observed phenotype. Whole-genome sequencing showed the MSSA to be multilocus sequence type (ST) 4371 (a single-locus variant of ST398). Sequencing also found no genes encoding superantigens (9 enterotoxins and This case of severe, widely disseminated infection in an immunocompetent man caused by a strain of MSSA without classic virulence factors is consistent with a growing body of evidence in support of MSSA CC398 as an emerging human pathogen. In cohorts of patients in France, CC398 MSSA has increased from being found in no cases in 1999 to 4.6% of cases in 2010 and 13.8% of cases in 2014 (A study in New York, New York, USA, found MSSA CC398 infection to be associated with a largely Dominican neighborhood, in particular; Hispanic ethnicity was a clinical predictor (Existing literature demonstrates evidence for MSSA CC398 as both a community- and hospital-associated pathogen (This case report aligns with existing evidence for MSSA CC398 as an emerging human pathogen. However, it is unusual in its degree of severity, with multiple extensive foci of infection, despite this strain lacking classic virulence factors. This lineage is of increasing global public health concern, and potential unidentified virulence factors and uncharacterized transmission patterns remain to be determined.


Due to the inevitable presence of random defects, unpredictable grain boundaries in macroscopic samples, stress concentration at clamping points, and unknown load distribution in the investigation of graphene sheets, uncertainties are crucial and challenging issues that require more exploration. The application of the Kriging surrogate model in vibration analysis of graphene sheets is proposed in this study. The Latin hypercube sampling method effectively propagates the uncertainties in geometrical and material properties of the finite element model. The accuracy and convergence of the Kriging surrogate model are confirmed by a comparison with the reported references. The uncertainty analysis for both Zigzag and Armchair graphene sheets are compared and discussed. With a two-dimensional 2D) honeycomb lattice, graphene can be wrapped up into zero-dimensional (0D) fullerenes, rolled into one-dimensional 1D) nanotubes, or stacked into three dimesnional 3D) graphite [D nanotubD honeycoD graphitIt is recognized in academia that graphene has the astonishing strength and stiffness when compared with the traditional materials. However, the precise values of the parameters corresponding to the material properties of graphene are difficult to determine. On the aspects of experimental measurements, the in-plane Young’s modulus of bulk graphite  is in thThe fluctuation and deviation in the parameters related with the material properties of graphene are due to the inevitable uncertainties. On the one hand, the nanoscale size in graphene ensures the exact measurement become challenging, and the relative errors caused by equipment or other stochastic factors are amplified and significantly affects the final results. On the other hand, the mathematical expressions and related knowledge in macro scale are limited and not appropriate to describe the physical relationships in microscope. The definition and evaluation of certain parameters in micro scale are required to be developed with accurate recognition.Different from epistemic uncertainties, the aleatory uncertainties in graphene are also unavoidable in the real situations. First, the random distributed defects contribute to the variation of material properties in graphene . Second,Development of sophisticated models to propagate the uncertainties in the deterministic models are vital issues in the mechanical analysis of graphene. The Latin hypercube sampling (LHS) method is one of the advanced Monte Carlo simulations (MCS). The MCS has been applied in the investigation of the phase transition and magnetism of graphene sheets ,15,16,17The Kriging surrogate model (KSM) is an interpolation method, which finds its roots in geo-statistics ,22. WithThis paper proposes the application of the KSM to represent the uncertain and complicated relationship between the elastic response of graphene sheets and the external forces. The LHS method is combined with the finite element model to successfully propagate the uncertainties in parameters corresponding to material and geometrical properties. The accuracy and convergence are confirmed by comparison with the reported references. The uncertainty analysis of Zigzag and Armchair graphene sheets in free vibration is completed and discussed.The modified Morse potential is successfully employed to simulate the nonlinear response of nanomaterials under tensile and torsional loading conditions . The effIn the finite element model of graphene sheets, the length of the C-C bond corresponds to the length of the beam in the planar-frame structure, while the wall thickness is related to the diameter of the cross section in the beam elements. However, the KSM prosposed in this study for uncertainty analysis of graphene based on the finite element method is different with the Monte Carlo based finite element method in the previous work. First, the motivation of KSM is for the uncertainty analysis of pristine graphene. While the Monte Carlo-based finite element method (MC-FEM) is to propagate the vacancy defects in the pristine graphene. Second, the research objects are different. The MC-FEM is more concentrated in the geometrical uncertainties such as vacancy defects. The study in this paper is more comprehensive to explore the uncertainties in the material and geometrical parameters. Third, compared with MC-FEM, the KSM in this paper has the following advantages: compatibility to the experimental data, convenience to the reliable prediction, and good performance in vibration analysis and uncertainty analysis for graphene. The MC-FEM is an effective numerical simulation to take the randomly distributed vacancy defects into consideration. However, the MC-FEM is not compatible to the experimental data or act as a surrogate model to represent the relationships between the input materials and geometrical parameters with the output resonant frequencies as well as their variance and fluctuations. Lastly, besides the vacancy defects in the graphene, the uncertainties and fluctuations in the material and geometrical parameters are also important issues to deal. The work in this study is a crucial supplement for the mechanical research of graphene.The mathematical description of the KSM is written as the following equation.variance .(2)Cov[The correlation kernel is usually described by a function of the distance.The weighted distance is often written as the following formula.Then, if the correlation kernel The correlation kernel is important to the performance of the KSM model. The common spatial correlation kernels are the Linear, Exponential, Gaussian, and Power-exponential . Among tFor the Universal Kriging (UK), the mean value of the prediction result for the input point The variance of the predictor A special case of UK is the Ordinary Kriging (OK). The trend is an unknown constant In the Simple Kriging (SK), the trend is a known constant value The LHS method is one kind of advanced Monte Carlo simulations (MCS). By dividing the range of each variable into disjointed intervals with equal probabilities, the samples are randomly selected from each interval in LHS. It improves the stability of MCS and keeps satisfied accuracy and good convergence ,19. ConsThe LHS method divides the range of each vector component into disjointed subsets with equal probabilities. Samples of each vector component are captured from the respective subsets, according to Equation (17).In order to demonstrate the KSM for graphene sheets, The blue boxes represent the procedure of the deterministic finite element model for vibration analysis of graphene sheets. First, the geometrical configuration of graphene sheets is defined. The corresponding parameters of bonds’ length and sectional diameter, as well as the height and width of a hexagonal 2D lattice are settled. Next, material parameters, which include the Young’s module, Poisson ratio, and physical density are provided. Then, perform the finite element model to compute the vibration modes and related natural frequencies.After validation of the finite element model, the loop performs until sufficient times of sampling are completed, as presented in the red boxes. In the continuous loop, parameters corresponding to geometrical and material properties are the input variables of KSM, while the natural frequencies of the free vibration by finite element analysis are also captured and transferred to the procedure of KSM. These two groups of databases form sampling pairs in KSM.In the pink boxes, the input variables of LHS and the output results of the finite element method (FEM) are sample pairs in KSM. In order to obtain satisfied prediction accuracy and good convergence, regression and correlation functions are required. The prediction results of KSM can be confirmed by comparison with the results in the reported references.The numerical simulation model of graphene sheets was created by the ANSYS Parameter Design Language . Carbon atoms in graphene are bonded together with covalent bonds, which form a hexagonal 2D lattice. The displacement of individual atoms under an external force is constrained by the bonds. For the modeling of the C-C bonds, the 3D elastic BEAM188 element was used. Each node has six degrees of freedom. The translations and rotations around the x, y, and z directions are included. In In the deterministic beam finite element model (FEM), there are 6226 beams with 16,664 nodes. By LHS, the stochastic values for each parameter in the certain intervals are sampled for the finite element model of graphene sheets.As mentioned above, the uncertainties in the geometrical and material properties are propagated in the finite element model by LHS. Furthermore, 1000 samples for each input parameter and output result are obtained in the computational process. Based on the original database, the results are studied by the stochastic mathematics and probability theory, as shown in In The cumulative probability of the first four order natural frequencies is compared in Even though all the input variables  of LHS are uniformly distributed in the sample space, the output results  do not obey the uniform or strict normal distribution, as shown in The KSM is applied to predict the natural frequencies of graphene sheets. Comparison between the prediction results of KSM and that of the reported References ,47,48,49In The prediction results of KSM are all larger than that of Sadeghzadeh  in the fIn In addition, the results in this study are in good agreement with that of nonlocal plate model proposed by Ansari . DiffereThe competitive predicition competence in KSM is not only applied in deterministic models, but can be used in uncertainty analysis. Based on the satisfied accuracy and robust convergence, KSM is applied in the uncertainty analysis of geometrical and material properties in graphene sheets.In In addition, for the uncertainties in material properties in Compared with the finite element method compuation, the advantage of time-saving in the KSM is presented in the uncertainty analysis process of graphene sheets. The time costs of natural frequencies and the vibration mode calcuation for each deterministic sample of graphene by finite element methods are nearly 5.6 s. However, based on the original database of LHS from the finite element model of graphene, the KSM can be applied to predict the natural frequencies with high efficiency. For the predicition of 1000 samples of graphene, it only takes approximated 12.3 s by KSM. When the parameters corresponding to geometrical and material properties are randomly distributed, performing the finite element model for sufficient times are computationally expensive and time consuming. Thus, the advantage of time-saving in the Kriging surrogate model compared with the traditional finite element model is evident.In addition, even though the exact mathematical relationship or functional expression between the graphene size and natural frequencies are not explicit, the KSM is proposed to effectively describe the implicit relationship. Furthermore, the KSM is compatible for the database of the numerical simulation and experimental tests. It is possible to combine the results of numerical simulation and physical experiments and create a more inclusive and smart database. Therefore, developing the KSM in the study of graphene sheets and related nanomaterials are promising and crucial.In summary, the Kriging surrogate model is not only a powerful method with satisfied accuracy and convergence in prediction, but also convenient and time-saving in uncertainty analysis of graphene sheets. The difficulties in uncertainty and vibration analysis of graphene sheets are well overcome by the Kriging surrogate model based on the LHS. With the comprehensive uncertainties in geometrical and material properties, the probabilty results of the LHS illustrate that the concentrated narrow interval with a long drag section is more appropriate than the strict normal probability density distribution. Furthermore, Zigzag graphene sheets are more robust and less senstive to the uncertainties of the geometrical property than Armchair graphene sheets in the free vibration. However, the uncertainties in materical properties cause larger fluctuation in Zigzag graphene sheets than in Armchair graphene sheets.


C9ORF72 expansion is the most common genetic alteration in both familial and sporadic ALS [C9ORF72 transcripts and may be toxic to cells [C9ORF72-associated amyotrophic lateral sclerosis (C9ALS), being identified in small neurons of hippocampus and cerebellum, as well as in neocortical neurons [to cells , 14 in ato cells . DPRs in neurons  and rare neurons . DPRs ma neurons  and are  neurons .C9ORF72 model demonstrated DPRs in skeletal muscle [C9ORF72 model showed distinct perinuclear poly-GP inclusions in muscle [Several studies have also suggested that DPRs are not limited to neurons. One study of human tissue, applying antibodies against GGGGCC RAN-translated peptides, showed DPRs predominantly in neurons, but also in Sertoli cells . Subsequl muscle , as welln muscle . This suTo address this question, we examined DPR inclusion pathology in 68 C9ALS skeletal muscle samples obtained from autopsies at Houston Methodist Hospital (HMH) and Mayo Clinic Jacksonville (MCJ). We assessed samples for the most common DPRs in human disease , poly-GR, as well as p62, N-terminal TDP-43, and phospho-TDP-43 (pTDP-43) pathology, using previously described methods  . Ultrastructural evaluation of a DPR-positive sample showed homogeneous intranuclear material and haphazardly arranged tubule-like structures in close proximity to subsarcolemmal nuclei. These ultrastructural findings were not seen in non-C9ALS postmortem muscle samples; however, DPRs were too sparse to definitively identify without immunoelectron microscopy, which will be a focus of future studies. Phospho-TDP-43 inclusions, also shown by N-terminal TDP-43, were present in 22 samples (32.4%) from 17 patients (46.0%) and co-localized with p62 . TDP-43-positive foci were uniformly negative for phospho-tau and beta-amyloid and their ultrastructural correlates have previously been shown [DPRs were identified in 28 of 68 samples 41.2%) from 18 of 37 patients (48.6%). Figure .2% from Z = 4.4, P = 1.12E − 05) . DPR pathology did not significantly associate with age at death, disease duration, or pTDP-43 pathology. Among samples with any inclusion pathology, 40% of patients had DPRs and pTDP-43, 32% had DPRs only, and 28% had pTDP-43 only. DPR-positive muscle samples were from patients with lower extremity (44.4%), upper extremity (22.2%) and bulbar (22.2%) onset sites. pTDP-43-positive and negative muscle samples did not significantly differ with respect to disease duration (P = 0.37), though a trend was seen for pTDP-43 pathology in younger C9ALS patients (P = 0.06). DPR-positive and pTDP-43-positive samples did not significantly differ in frequency by axial muscle group.DPRs were significantly associated with more severe atrophy in samples . RAN-translated peptides were not seen in three patient muscle samples in a prior study . A separxpansion . Future Supplementary file1 (PDF 960 kb)Below is the link to the electronic supplementary material.


Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions.The entomopathogenic fungus M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis.Neem was mixed with Laboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation.M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.A combination of the entomopathogenic fungus The online version of this article (10.1186/s13071-019-3415-x) contains supplementary material, which is available to authorized users. Aedes aegypti, namely dengue, Zika, chikungunya and urban yellow fever, are of growing concern, not only in tropical and semi-tropical regions of the world, but also in southern Europe where it is now a threat  was placed in 700 ml black rectangular plant pots  were suspended in Tween 80 (TW 80) (0.05% v/v) and subsequently neem oil was added to a final concentration of 1% and 0.01% (v/v). Fungal suspensions were also prepared in TW 80 without neem. Three milliliters of each sample was placed in plastic Petri dishes (55 mm diameter) and exposed to a UV-B lamp , which at a distance of 70 cm was equivalent to a UV-B intensity of 1630 mW m-2. Conidia were exposed to UV radiation for 0.5, 1, 2, 3, 4 and 5 h periods. Controls for each treatment were not exposed to UV light.These experiments were carried out under laboratory conditions with the aim of investigating the possibility that neem oil protects conidia from the deleterious effects of UV radiation. Conidia  and incubating at 27 °C (12 h light: 12 h dark regime) for 12 h. At this time the number of conidia germinating was quantified using an optical microscope . Three fields of view were counted for each sample by the same researcher (AR). Conidia were considered as germinated when the germ tube was of equal size or larger than the conidia itself. Each treatment was replicated three times.post-hoc test at the 5% level was used.Mean survival rates were compared using one-way analysis of variance. When significant differences were seen between treatments or between time points within the same treatment, the data was further analyzed by Duncan’s post hoc test at the 5% level. The persistence/half-life of the different treatments was evaluated by using polynomial regression analysis, with the effective half-life given as the time (in days) that the treatments resulted in 50% larval survival/mortality. Differences in mean germination rates following exposure to UV were compared using one-way analysis of variance. When significant differences were detected, Duncan’s 9 conidia ml-1) and a combination of neem + fungus, with larvae exposed to the different treatments immediately following preparation (time zero) or following different time periods of up to 50 days. The results for these experiments are shown in Table Ae. aegypti larvae when exposed to the fungus alone was 30% at time zero , whilst for neem alone the larval survival rate was 74% at time zero. However, exposure of larvae to the formulation fungus + neem resulted in 11% survival at time zero. Fungus + neem significantly reduced survival rates when compared to the other two treatments and to the controls  = 1446.2, P < 0.01). The survival rates for larvae exposed to the fungus alone were not significantly different (P > 0.01) to the controls following 10 days under laboratory conditions, showing the low half-life of the fungus when suspended in TW 80. The slight but significant reduction in survival seen at time zero for neem alone was transitory, with no significant reduction in survival by day 5 (81%) when compared to controls (83%).Before testing fungal persistence in the field, bioassays were carried out under laboratory conditions using 0.01% neem (v/v), fungus  = 157.5, P < 0.01). When larvae were exposed to this formulation following 50 days in recipients, the survival rate was 74%, which was not significantly different to the controls (P > 0.01). The half-life of this formulation under laboratory conditions was 42.8 days as calculated by regression analysis .The survival rates were then monitored for the formulation fungus + neem maintained in recipients for different periods of up to 50 days. The mixture of fungus + neem continued to significantly reduce larval survival rates when maintained in recipients for up to 45 days, with 58% survival observed at this time (P > 0.01) following 10 days of maintaining the fungus or neem oil under field conditions  = 236.9, P < 0.01), but larvicidal activity was rapidly lost. Therefore, the next set of experiments was carried out using 0.1% (v/v) neem.These tests were similar to those described above but with recipients maintained under field conditions. Two preliminary experiments were carried out but both were stopped after 10 days. The first experiment was set up using the same concentration of neem as that used in laboratory experiments, 0.01% (v/v). However, none of the treatments were significantly different to the controls  = 441.0, P < 0.01), the experiment was stopped due to the rapid decline in larvicidal activity.However, a similar pattern was seen for the experiments using 0.1% neem, with larvicidal activity rapidly reduced after 10 days under field conditions  = 478.4, P < 0.01). At time zero, the fungus alone significantly reduced larval survival  when compared to the controls, as did neem alone , with no significant difference between these two treatments  neem  = 556.6, P < 0.01). Fifty days after maintaining this formulation under simulated field conditions, and then adding larvae to the recipients, fungus + neem was no longer effective at reducing survival rates and there was no significant difference between the treatments.The persistence of the formulations was evaluated for up to 50 days under simulated field conditions. When comparing survival rates of all treatments with controls over time, the formulation fungus + neem maintained significant larvicidal activity until day 45 , at which time the survival rates were statistically different to the other treatments and the controls (P > 0.01). Neem alone caused significant reductions in survival (72%) until day 25 when compared to the controls.The fungus when tested alone under field conditions rapidly lost virulence, with no difference to the controls at 25 days, when survival rates were 83%  = 46.07, P < 0.01), with 80.3% (± 1.69%) germination observed. However, 3 to 5 h exposures caused significant reductions in germination when compared to shorter exposure times. Neem 0.01% (v/v) was a less effective protectant, with rapid and significant reductions in conidial germination when exposing conidia to UV-B for 1 h or more  = 9270.0, P < 0.01). All treatments showed low levels of germination following a 5-h exposure to UV. The mean germination rates of all three control treatments were not significantly different (P > 0.01) and approximately 80% of the conidia had germinated when quantified 12 h after inoculating the suspensions in culture media. The complete data and statistical analysis for the germination experiments are shown in Additional file The results for the three treatments investigated here showed the significant protective characteristics of neem when considering the deleterious effects of UV radiation on conidial germination Fig. . When coF5,7 = 46.07Bacillus thuringiensis serotype israelensis (Bti). When Bti is applied in the field in recipients exposed to sunlight, control is efficient for up to four weeks. However, when used in recipients not directly exposed to sunlight, the product is effective for up to 11 weeks [Current conventional mosquito larval control measures in domestic environments involves local government agents visiting residences and treating putative breeding sites, such as drains and water tanks with synthetic chemical larvicides or products containing 11 weeks .Metarhizium conidia in mineral oil in a product known as Green Muscle®, used for the control of the desert locust [One of the most serious drawbacks of deploying entomopathogenic fungi for pest and vector control is their short effective half-life under field conditions. With the aim of increasing the persistence of these fungi, many studies have been carried out on different formulation technologies . Amongstt locust . In thist locust .M. anisopliae against Ae. aegypti larvae [In our previous study of neem oil as an adjuvant for use with entomopathogenic fungi, very low concentrations of neem oil significantly increased the virulence of i larvae , probablAe. aegypti larvae were exposed to neem oil, their development was affected, resulting in a significant reduction in adult emergence [An. stephensi and An. gambiae were reduced following exposure to neem. Pupal formation and adult emergence were significantly reduced in the case of An. stephensi and An. gambiae larvae exposed to neem [Neem oil has insecticidal properties and is currently used for pest control in organic farming systems . Howevermergence , 25. Fee to neem , 27.Ae. aegypti larvae. Neem when used at 0.01% (v/v) together with M. anisopliae enabled the fungus to significantly reduce larval survival rates for up to 45 days after the suspension had been prepared and maintained on the bench. The fungus when used alone rapidly lost virulence, with survival rates equal to that of the controls after only 10 days. The decline in virulence of M. anisopliae seen here was greater than that seen previously, when the half-life of the same fungus was estimated to be 10 days under similar conditions [In the first set of experiments here, under laboratory conditions, the concentration of neem that increased fungal persistence was not considered to be toxic to nditions .Ae. aegypti oviposition sites. The same type of plant pot is normally used as a so called “ovitrap” to collect eggs in the field. Although in the present experiments, mosquito larvae were added to the pots to test the persistence of the formulations, future experiments will be carried out using ovitraps with fungus + neem in the field as a possible control strategy against the egg stage and any larvae emerging from the eggs. However, we have yet to test if this formulation could be detected by females and act as a repellant to oviposition. Studies have shown that Ae. aegypti females are not repelled by the presence of M. anisopliae conidia on black cloths [An. gambiae, which oviposit higher numbers of eggs in the presence of bacteria than in sterile water [The persistence of the fungus when formulated with neem oil was tested under field conditions. The present field experiments were performed using black plant pots, which are highly suitable as k cloths ; howeverle water .Ae. aegypti larvae. Neem at 1% (v/v) has been previously shown to cause a 50% reduction in survival rates within two days [An. stephensi larvae [Ae. aegypti larvae when used alone, the advantage of formulating M. anisopliae with this concentration of neem was a significant increase in the fungal half-life in the field. The half-life of the fungus alone was only six days, which would seriously impair its use under field conditions. The larvicidal activity of neem also fell rapidly under these conditions, with an estimated half-life of 13 days seen here. This was probably due to bio-degradation.The initial results for field tests , exposure of conidia to UV rapidly reduced viability. However, conidial suspensions in 1% neem oil protected the conidia for up to two hours exposure to UV, with no significant reductions in germination when compared to controls. For sesame or colza oil, the protective capacity was approximately 70% following a five-hour exposure to UV-B following field trails .M. anisopliae. This work shows for the first time an important strategy for maintaining fungal virulence over extended time periods. One of the major drawbacks of using entomopathogenic fungi is their short half-life. However, the addition of neem to conidial suspensions not only increased virulence, but significantly increased the persistence of the fungus.Neem was tested in the laboratory and under field conditions as an adjuvant for the entomopathogenic fungus Additional file 1: Figure S1. Black rectangular plant pot used for field testing the virulence and persistence of the fungal and neem oil treatments against Ae. aegypti larvae. Figure S2. Field testing the persistence of the conidia against Ae. aegypti larvae. Experiments on a covered veranda were carried out within netting cages to prevent mosquitoes using the plant pots as oviposition sites. Table S1. Survival rates of Ae. aegypti larvae following exposure to different treatments under simulated field conditions. Table S2. Survival rates of Ae. aegypti larvae following exposure to different treatments under simulated field conditions. Table S3. Germination rates (mean % ± SD) of Metarhizium anisopliae conidia following exposure to UV-B when formulated in two concentration of neem oil or Tween.