Rapid diagnosis of bloodstream infections using a culture-free phenotypic platform.

BACKGROUND: Bloodstream infections (BSIs) are a life-threatening acute medical condition and current diagnostics for BSIs suffer from long turnaround time (TAT). Here we show the validation of a rapid detection-analysis platform (RDAP) for the diagnosis of BSIs performed on clinical blood samples METHODS: The validation was performed on a cohort of 59 clinical blood samples, including positive culture samples, which indicated confirmed bloodstream infections, and negative culture samples. The bacteria in the positive culture samples included Gram-positive and Gram-negative pathogenic species. RDAP is based on an electrochemical sandwich immunoassay with voltage-controlled signal amplification, which provides an ultra-low limit of detection (4 CFU/mL), allowing the platform to detect and identify bacteria without requiring culture and perform phenotypic antibiotic susceptibility testing (AST) with only 1-2 h of antibiotic exposure. The preliminary diagnostic performance of RDAP was compared with that of standard commercial diagnostic technologies. RESULTS: Using a typical clinical microbiology laboratory diagnostic workflow that involved sample culture, agar plating, bacteria identification using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry, and AST using MicroScan as a clinical diagnostic reference, RDAP showed diagnostic accuracy of 93.3% and 95.4% for detection-identification and AST, respectively. However, RDAP provided results at least 15 h faster. CONCLUSIONS: This study shows the preliminary feasibility of using RDAP to rapidly diagnose BSIs, including AST. Limitations and potential mitigation strategies for clinical translation of the present RDAP prototype are discussed. The results of this clinical feasibility study indicate an approach to provide near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.

Effective treatment of bloodstream infections (BSIs), a life-threatening acute medical condition, requires rapid diagnosis. Current diagnostic methods involve culturing the bacteria from the patient's blood, which requires typically 16­48 h to produce a diagnosis. Here, we demonstrate the feasibility of using a culture-free platform to perform rapid diagnosis of BSIs. We tested the diagnostic platform on a cohort of clinical blood samples. The bacteria contained in the samples covered a representative range of bacteria that cause BSIs. The culture-free platform produced diagnosis in about 15 hours faster than standard commercial diagnostic technologies and  the diagnostic results were in good agreement with that of standard technologies. The results of this study indicate an approach to providing near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.

Nodeless Superconductivity in Kagome Metal CsV3Sb5 with and without Time Reversal Symmetry Breaking.

The kagome metal CsV3Sb5 features an unusual competition between the charge-density-wave (CDW) order and superconductivity. Evidence for time reversal symmetry breaking (TRSB) inside the CDW phase has been accumulating. Hence, the superconductivity in CsV3Sb5 emerges from a TRSB normal state, potentially resulting in an exotic superconducting state. To reveal the pairing symmetry, we first investigate the effect of nonmagnetic impurity. Our results show that the superconducting critical temperature is insensitive to disorder, pointing to conventional s-wave superconductivity. Moreover, our measurements of the self-field critical current (Ic,sf), which is related to the London penetration depth, also confirm conventional s-wave superconductivity with strong coupling. Finally, we measure Ic,sf where the CDW order is removed by pressure and superconductivity emerges from the pristine normal state. Our results show that s-wave gap symmetry is retained, providing strong evidence for the presence of conventional s-wave superconductivity in CsV3Sb5 irrespective of the presence of the TRSB.

Patterned diamond anvils prepared via laser writing for electrical transport measurements of thin quantum materials under pressure.

Quantum materials exhibit intriguing properties with important scientific values and huge technological potential. Electrical transport measurements under hydrostatic pressure have been influential in unraveling the underlying physics of many quantum materials in bulk form. However, such measurements have not been applied widely to samples in the form of thin flakes, in which new phenomena can emerge, due to the difficulty in attaching fine wires to a thin sample suitable for high-pressure devices. Here, we utilize a home-built direct laser writing system to functionalize a diamond anvil to directly integrate the capability of conducting electrical transport measurements of thin flakes with a pressure cell. With our methodology, the culet of a diamond anvil is equipped with a set of custom-designed conducting tracks. We demonstrate the superiority of these tracks as electrodes for the studies of thin flakes by presenting the measurement of pressure-enhanced superconductivity and quantum oscillations in a flake of MoTe2.

Detection of a Traumatic Brain Injury Biomarker at the 10 fg/mL Level.

BACKGROUND: The detection of minute amounts of protein biomarkers in body fluids is believed to provide early diagnosis and prognosis of mild traumatic brain injury (mTBI). An ultrasensitive detection method was used to detect S100B, the most studied potential marker for the diagnosis of mTBI. METHODS: The detection method was a modified electrochemical immunoassay technique that provides voltage controlled intrinsic current signal amplification. The sandwich immune complex of S100B was formed on the working electrode of the screen-printed electrode. The gating voltage provides amplification of the current signal that flows through the complex. RESULTS: S100B was spiked in human serum. The limit of detection of S100B in human serum was 10 fg/mL. The calibration curves cover four orders of magnitudes from 10 fg/mL to 10 ng/mL. The specificity of the detection was demonstrated using TAU protein, which is another marker for mTBI. CONCLUSION: The results reported in this work using the field effect enzymatic detection (FEED)-based immunoassay indicate the feasibility of using this method for the detection of extremely low concentrations of markers of mTBI in human serum. This method can be developed as a platform for a range of markers of mTBI.

Culture-free bacterial detection and identification from blood with rapid, phenotypic, antibiotic susceptibility testing.

The current culture-based approach for the diagnosis of bloodstreams infection is incommensurate with timely treatment and curbing the prevalence of multi-drug resistant organisms (MDROs) due to its long time-to-result. Bloodstream infections typically involve extremely low (e.g., <10 colony-forming unit (CFU)/mL) bacterial concentrations that require a labor-intensive process and as much as 72 hours to yield a diagnosis. Here, we demonstrate a culture-free approach to achieve rapid diagnosis of bloodstream infections. An immuno-detection platform with intrinsic signal current amplification was developed for the ultrasensitive, rapid detection, identification (ID) and antibiotic susceptibility testing (AST) of infections. With its capability of monitoring short-term (1-2 hours) bacterial growth in blood, the platform is able to provide 84-minute simultaneous detection and ID in blood samples below the 10 CFU/mL level and 204-minute AST. The susceptible-intermediate-resistant AST capacity was demonstrated.

Declining Trends of Cardiovascular-Renal Complications and Mortality in Type 2 Diabetes: The Hong Kong Diabetes Database.

OBJECTIVE: Nationwide studies on secular trends of diabetes complications are not available in Asia. We examined changes in risk factor control and incidence of complications from diabetes and death in a large longitudinal cohort of Chinese adults with type 2 diabetes in Hong Kong. RESEARCH DESIGN AND METHODS: Between 1 January 2000 and 31 December 2012, 338,908 Chinese adults with type 2 diabetes underwent metabolic and complication assessment in 16 diabetes centers operated by Hong Kong Hospital Authority that provided care to a large majority of diagnosed patients. Patients were followed for incident acute myocardial infarction (AMI), stroke, end-stage renal disease (ESRD), and death until 31 December 2012. Risk factor levels between enrollment periods were compared. Incidence of clinical events, stratified by diabetes duration, was examined over time. RESULTS: Incidence of complications from diabetes and death declined over the observation period in patients at varying disease duration. Among the high-risk group with diabetes for at least 15 years, crude incidence of AMI decreased from 8.7 to 5.8, stroke from 13.5 to 10.1, ESRD from 25.8 to 22.5, and death from 29.0 to 26.6 per 1,000 person-year between the periods 2000 to 2002 and 2010 to 2012. Improvements in levels of metabolic risk factors were detected. Proportion of patients achieving HbA1c <7.0% (53 mmol/mol) was increased from 32.9 to 50.0%, blood pressure ≤130/80 mmHg from 24.7 to 30.7%, and LDL cholesterol <2.6 mmol/L from 25.8 to 38.1%. CONCLUSIONS: From this territory-wide Hong Kong Diabetes Database, we observed decreases in incidence of cardiovascular-renal complications and death and corresponding improvements in risk factor control over a 13-year period.

Ultrasensitive Detection of Shigella Species in Blood and Stool.

A modified immunosensing system with voltage-controlled signal amplification was used to detect Shigella in stool and blood matrixes at the single-digit CFU level. Inactivated Shigella was spiked in these matrixes and detected directly. The detection was completed in 78 min. Detection limits of 21 CFU/mL and 18 CFU/mL were achieved in stool and blood, respectively, corresponding to 2-7 CFUs immobilized on the detecting electrode. The outcome of the detection of extremely low bacterium concentration, i.e., below 100 CFU/mL, blood samples show a random nature. An analysis of the detection probabilities indicates the correlation between the sample volume and the success of detection and suggests that sample volume is critical for ultrasensitive detection of bacteria. The calculated detection limit is qualitatively in agreement with the empirically determined detection limit. The demonstrated ultrasensitive detection of Shigella on the single-digit CFU level suggests the feasibility of the direct detection of the bacterium in the samples without performing a culture.

Enhanced ethanol production via electrostatically accelerated fermentation of glucose using Saccharomyces cerevisiae.

The global demand for ethanol as an alternative fuel continues to rise. Advancement in all aspects of ethanol production is deemed beneficial to the ethanol industry. Traditional fermentation requires 50-70 hours to produce the maximum ethanol concentration of 7-8% (v/v). Here we demonstrate an electrostatic fermentation method that is capable of accelerating the fermentation of glucose using generic Saccharomyces cerevisiae as the fermenting microorganism to produce ethanol. The method, when applied to the batch fermentation of 1 liter fermenting mixture containing dry yeast without pre-culture, is able to achieve ethanol yield on the high gravity level (12.3% v/v) in 24 hours. The fermentation results in almost complete consumption of glucose. With pre-cultured yeast, ethanol yield can reach 14% v/v in 20 hours. The scale-up capability of the method is demonstrated with 2 liter fermenting mixture. The method does not consume external energy due to its electrostatic nature. Our results indicate the applicability of the fermentation technique to industry applications.

Controlled glucose consumption in yeast using a transistor-like device.

From the point of view of systems biology, insight into controlling the functioning of biological systems is conducive to the understanding of their complexness. The development of novel devices, instrumentation and approaches facilitates this endeavor. Here, we show a transistor-like device that can be used to control the kinetics of the consumption of glucose at a yeast-immobilised electrode. The gating voltage of the device applied at an insulated gating electrode was used to control both the rate of glucose consumption and the rate of the production of ATP and ethanol, the end-products of normal glucose metabolism. Further, a correlation between the glucose consumption and the production of ethanol controlled by the gating voltage was observed using two different forms of the device. The results suggest the relevance of glucose metabolism in our work and demonstrate the electrostatic nature of the device. An attempt to explain the effect of the gating voltage on the kinetics is made in terms of transfer of electrons from NADH to enzymes in the electron transport chain. This novel technique is applicable to general cells and the reported results show a possible role for electrostatic means in controlling processes in cells.

Voltage-controlled enzyme-catalyzed glucose-gluconolactone conversion using a field-effect enzymatic detector.

The field-effect enzymatic detection (FEED) technique was used to control the kinetics of the enzymatic conversion of glucose to gluconolactone. The glucose-gluconolactone conversion occurring at an enzyme-immobilized electrode, a well-studied process, was confirmed using mass spectrometry. Electrochemical studies showed that the glucose oxidation current depends on the gating voltage VG and the ion concentration of the sample solution. Additionally, the depletion of glucose in the sample also showed a dependence on VG. FEED was used to detect H2O2 on the zepto-molar level in order to show the ultrasensitive detection capability of the technique. These results, while providing evidence for the proposed mechanism of FEED, indicate that VG controls the conversion process. The effect of VG on the glucose-gluconolactone conversion was demonstrated by the observed VG-dependent kinetic parameters of the conversion process.

Bedside ultrasonography performed by family physicians in outpatient medical offices in Whitehorse, Yukon.

INTRODUCTION: We sought to determine the current practices and opinions of family physicians in Whitehorse, YT, regarding bedside ultrasonography performed by family physicians in outpatient medical offices. METHODS: A paper survey was administered to Whitehorse family physicians. Only those who had worked for longer than 6 months in a community outpatient clinic in Whitehorse were invited to participate. RESULTS: The response rate of our survey was 44%. None of the respondents reported currently using bedside ultrasonography in their outpatient medical offices; however, 78% reported having training in ultrasonography and using it in another setting. Of the respondents, 94% stated they would consider using bedside ultrasonography in their outpatient medical office. Economics was the biggest reported barrier in the use of bedside ultrasonography in outpatient medical offices. CONCLUSION: A wealth of experience in bedside ultrasonography already exists among family physicians in Whitehorse, and an overwhelming majority of physicians are ready to embrace its use in outpatient offices. However, the skills and willingness of family physicians have not translated into the use of bedside ultrasonography in outpatient medical offices.

Field-effect amperometric immuno-detection of protein biomarker.

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work.

Increased accumulation of paclitaxel and doxorubicin in proliferating capillary cells and prostate cancer cells following ultrasound exposure.

INTRODUCTION: We have previously reported enhanced cytotoxic effects of both doxorubicin and antisense oligonucleotides using an optimized ultrasound regime of a single 10s exposure in burst-mode (4 MHz, 32 W/cm(2)(SaTa), 50 ms burst period) in both PC3 (prostate cancer) cells and angiogenic Huvec (human umbilical cord endothelial cells). The objective of this study was to investigate the effect of ultrasound on the cellular uptake of both hydrophilic agents (rhodamine R123, doxorubicin hydrochloride and mannitol) and hydrophobic agents (rhodamine R6G and paclitaxel) using the same 4 MHz ultrasound exposure system. METHODS: PC3 cells and Huvec were incubated with solutions of radioactive or fluorescent compounds for 1h and ultrasound was then applied to cells. Following washing and lysis of cells, the degree of drug uptake was measured using liquid scintillation counting or fluorescence spectroscopy. RESULTS: Ultrasound exposure resulted in the enhanced uptake of both hydrophilic and hydrophobic compounds into cells. For paclitaxel, approximately 100% increased uptake was observed when the drug was encapsulated in a nanoparticulate micellar formulation compared to approximately 50% for free drug. CONCLUSIONS: The 4 MHz, 32 W/cm(2) ultrasound exposure regime (using burst-mode with 50 ms burst period) allows for the enhanced uptake of both water soluble and insoluble compounds into proliferating cancer and angiogenic cells.

Ultrasensitive biosensing on the zepto-molar level.

Detection of analytes on the zepto-molar (10(-21) M) level has been achieved using a field-effect bio-detector. By applying a gating voltage to enzymes immobilized on the working electrode of the detector, amplification of the biocatalytic current was observed. The amplification is attributed to the modification of the tunnel barrier between the enzyme and the electrode by the gating voltage-induced electric field which exists at the solution-electrode interface. The detection was demonstrated with the glucose oxidase (GOx)-glucose and alcohol dehydrogenase (ADH)-ethanol biocatalytic systems. Glucose at zepto-molar level was detected with zepto-molar detection resolution. Equivalently, 30 glucose molecules present in the sample were detected and the detection system responded distinctively to the incremental change in the number of glucose molecules in unit of 30 molecules. The enzyme's biospecificity was also preserved in the presence of the applied field. We present possible processes that could give rise to the electrical charges required to produce the observed current level.

Synthesis, characterisation and photophysical study of alkynylrhenium(I) tricarbonyl diimine complexes and their metal-ion coordination-assisted metallogelation properties.

A series of alkynylrhenium(I) tricarbonyl diimine complexes has been synthesized and characterized. A blue shift of the intense low-energy MLCT absorption band in the visible region was observed upon coordination of Cu(I) or Ag(I). This class of complexes has been found to show rich thermotropic gelation behaviour upon Cu(I) or Ag(I) coordination with their morphology characterized by SEM. Their variable-temperature UV/Vis absorption and emission properties have also been studied. A blue shift in the MLCT absorption band and the switching on of luminescence were observed upon sol-gel transition.

Synthesis and characterization of luminescent rhenium(I) tricarbonyl diimine complexes with a triarylboron moiety and the study of their fluoride ion-binding properties.

A series of alkynylrhenium(I) tricarbonyl diimine complexes with a triarylboron moiety has been synthesized and characterized. The binding properties of the complex toward fluoride ions have been studied using electronic absorption and emission spectroscopy and were further supported by (19)F NMR binding experiments.

Field-effect enzymatic amplifying detector with picomolar detection limit.

Voltage-controlled amplification of the output current of an enzymatic detector has been demonstrated. By application of an external voltage between the gating electrode and the working electrode on which the enzyme glucose oxidase was immobilized, the biocatalytic output current of the detector was increased significantly, allowing the detection limit of glucose to be lowered from the millimolar level to the picomolar level. The current amplification could be reversibly controlled by the applied voltage. Application of this technique to the ethanol-alcohol dehydrogenase system showed similar results. The detection setup suggests that the output current is controlled by the electric field at the interface between the solution and the working electrode. The enzyme's biospecificity was preserved in the presence of the field. The detector, with its output current controlled by a voltage applied at a third electrode, behaves as a field-effect transistor, whose current-generating mechanism is the conversion of an analyte to a product using an enzyme as catalyst. In a broader sense, the operation of the detector shows a means for manipulating a redox enzymatic reaction.

A hybrid biofuel cell based on electrooxidation of glucose using ultra-small silicon nanoparticles.

The ultra-small silicon nanoparticle was shown to be an electrocatalyst for the electrooxidation of glucose. The oxidation appeared to be a first order reaction which involves the transfer of 1 electron. The oxidation potential showed a low onset of -0.4V vs. Ag/AgCl (-0.62 V vs. RHE). The particle was used as the anode catalyst of a prototype hybrid biofuel cell, which operated on glucose and hydrogen peroxide. The output power of the hybrid cell showed a dependence on the enzymes used as the cathode catalyst. The power density was optimized to 3.7 microW/cm(2) when horseradish peroxidase was replaced by microperoxidase-11 (MP-11). Comparing the output power of the hybrid cell to that of a biofuel cell indicates enhanced cell performance due to the fast reaction kinetics of the particle. The long-term stability of the hybrid cell was characterized by monitoring the cell voltage for 5 days. It appeared to that the robustness of the silicon particle resulted in more cell stability compared to the long-term performance of a biofuel cell.

Impact of routine immunization using meningococcal C conjugate vaccine on invasive meningococcal disease in British Columbia.

OBJECTIVES: (1) To examine trends in serogroup-specific invasive meningococcal disease (IMD) incidence associated with the protein-polysaccharide conjugate C vaccine (MCC) program in BC; (2) To assess for evidence of capsule switching and serogroup replacement; (3) To discuss whether recent data support modification of the current MCC program to include the quadrivalent protein-polysaccharide conjugate vaccine (MCV-4). METHODS: Information on IMD cases since 1998 were extracted from surveillance databases. Annual IMD incidence rates and corresponding three-year moving averages were calculated. Data management was performed using Microsoft Office Excel 2003. Time trends were analyzed using chi-square test for linear trend. RESULTS: For 2003-2006, no significant trends were found in rates of serogroup-specific or total IMD in the overall BC population. Among children <18 years, average annual incidence of serogroup-C IMD has declined with a downward trend (p=0.05). Median age of serogroup-C IMD increased from 16 years (2003) to 42 years (2006). No significant change in incidence rates of pediatric IMD from any non-C serogroup was detected. DISCUSSION: We document a decreasing trend of pediatric serogroup-C IMD and an increase in median age of serogroup-C IMD cases since 2003, most likely explained by protection from immunization. While the proportion of serogroup-Y IMD has increased, incidence rates of non-C vaccine-preventable IMD have not increased in BC. While incorporation of MCV-4 in routine childhood immunization is desirable to address the few residual cases of non-C vaccine-preventable IMD, it would take several decades to appreciate a benefit from a modified childhood program.