The Biologically Active Biopolymer Silk: The Antibacterial Effects of Solubilized Bombyx mori Silk Fibroin with Common Wound Pathogens.

Antibacterial properties are desirable in wound dressings. Silks, among many material formats, have been investigated for use in wound care. However, the antibacterial properties of liquid silk are poorly understood. The aim of this study is to investigate the inherent antibacterial properties of a Bombyx mori silk fibroin solution. Silk fibroin solutions containing ≥ 4% w/v silk fibroin do not support the growth of two common wound pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. When liquid silk is added to a wound pad and placed on inoculated culture plates mimicking wound fluid, silk is bacteriostatic. Viability tests of the bacterial cells in the presence of liquid silk show that cells remain intact within the silk but could not be cultured. Liquid silk appears to provide a hostile environment for S. aureus and P. aeruginosa and inhibits growth without disrupting the cell membrane. This effect can be beneficial for wound healing and supports future healthcare applications for silk. This observation also indicates that liquid silk stored prior to processing is unlikely to experience microbial spoilage.

Impact of silk hydrogel secondary structure on hydrogel formation, silk leaching and in vitro response.

Silk can be processed into a broad spectrum of material formats and is explored for a wide range of medical applications, including hydrogels for wound care. The current paradigm is that solution-stable silk fibroin in the hydrogels is responsible for their therapeutic response in wound healing. Here, we generated physically cross-linked silk fibroin hydrogels with tuned secondary structure and examined their ability to influence their biological response by leaching silk fibroin. Significantly more silk fibroin leached from hydrogels with an amorphous silk fibroin structure than with a beta sheet-rich silk fibroin structure, although all hydrogels leached silk fibroin. The leached silk was biologically active, as it induced vitro chemokinesis and faster scratch assay wound healing by activating receptor tyrosine kinases. Overall, these effects are desirable for wound management and show the promise of silk fibroin and hydrogel leaching in the wider healthcare setting.

Analytical validation of a multi-biomarker algorithmic test for prediction of progressive kidney function decline in patients with early-stage kidney disease.

BACKGROUND: The KidneyIntelX™ test applies a machine learning algorithm that incorporates plasma biomarkers and clinical variables to produce a composite risk score to predict a progressive decline in kidney function in patients with type 2 diabetes (T2D) and early-stage chronic kidney disease (CKD). The following studies describe the analytical validation of the KidneyIntelX assay including impact of observed methodologic variability on the composite risk score. METHODS: Analytical performance studies of sensitivity, precision, and linearity were performed on three biomarkers assayed in multiplexed format: kidney injury molecule-1 (KIM-1), soluble tumor necrosis factor receptor-1 (sTNFR-1) and soluble tumor necrosis factor receptor-2 (sTNFR-2) based on Clinical Laboratory Standards Institute (CLSI) guidelines. Analytical variability across twenty (20) experiments across multiple days, operators, and reagent lots was assessed to examine the impact on the reproducibility of the composite risk score. Analysis of cross-reactivity and interfering substances was also performed. RESULTS: Assays for KIM-1, sTNFR-1 and sTNFR-2 demonstrated acceptable sensitivity. Mean within-laboratory imprecision coefficient of variation (CV) was established as less than 9% across all assays in a multi-lot study. The linear range of the assays was determined as 12-5807 pg/mL, 969-23,806 pg/mL and 4256-68,087 pg/mL for KIM-1, sTNFR-1 and sTNFR-2, respectively. The average risk score CV% was less than 5%, with 98% concordance observed for assignment of risk categories. Cross-reactivity between critical assay components in a multiplexed format did not exceed 1.1%. CONCLUSIONS: The set of analytical validation studies demonstrated robust analytical performance across all three biomarkers contributing to the KidneyIntelX risk score, meeting or exceeding specifications established during characterization studies. Notably, reproducibility of the composite risk score demonstrated that expected analytical laboratory variation did not impact the assigned risk category, and therefore, the clinical validity of the reported results.

Derivation and validation of a machine learning risk score using biomarker and electronic patient data to predict progression of diabetic kidney disease.

AIM: Predicting progression in diabetic kidney disease (DKD) is critical to improving outcomes. We sought to develop/validate a machine-learned, prognostic risk score (KidneyIntelX™) combining electronic health records (EHR) and biomarkers. METHODS: This is an observational cohort study of patients with prevalent DKD/banked plasma from two EHR-linked biobanks. A random forest model was trained, and performance (AUC, positive and negative predictive values [PPV/NPV], and net reclassification index [NRI]) was compared with that of a clinical model and Kidney Disease: Improving Global Outcomes (KDIGO) categories for predicting a composite outcome of eGFR decline of ≥5 ml/min per year, ≥40% sustained decline, or kidney failure within 5 years. RESULTS: In 1146 patients, the median age was 63 years, 51% were female, the baseline eGFR was 54 ml min-1 [1.73 m]-2, the urine albumin to creatinine ratio (uACR) was 6.9 mg/mmol, follow-up was 4.3 years and 21% had the composite endpoint. On cross-validation in derivation (n = 686), KidneyIntelX had an AUC of 0.77 (95% CI 0.74, 0.79). In validation (n = 460), the AUC was 0.77 (95% CI 0.76, 0.79). By comparison, the AUC for the clinical model was 0.62 (95% CI 0.61, 0.63) in derivation and 0.61 (95% CI 0.60, 0.63) in validation. Using derivation cut-offs, KidneyIntelX stratified 46%, 37% and 17% of the validation cohort into low-, intermediate- and high-risk groups for the composite kidney endpoint, respectively. The PPV for progressive decline in kidney function in the high-risk group was 61% for KidneyIntelX vs 40% for the highest risk strata by KDIGO categorisation (p < 0.001). Only 10% of those scored as low risk by KidneyIntelX experienced progression (i.e., NPV of 90%). The NRIevent for the high-risk group was 41% (p < 0.05). CONCLUSIONS: KidneyIntelX improved prediction of kidney outcomes over KDIGO and clinical models in individuals with early stages of DKD.

Toward a Closed Loop, Integrated Biocompatible Biopolymer Wound Dressing Patch for Detection and Prevention of Chronic Wound Infections.

Chronic wound infections represent a significant burden to healthcare providers globally. Often, chronic wound healing is impeded by the presence of infection within the wound or wound bed. This can result in an increased healing time, healthcare cost and poor patient outcomes. Thus, there is a need for dressings that help the wound heal, in combination with early detection of wound infections to support prompt treatment. In this study, we demonstrate a novel, biocompatible wound dressing material, based on Polyhydroxyalkanoates, doped with graphene platelets, which can be used as an electrochemical sensing substrate for the detection of a common wound pathogen, Pseudomonas aeruginosa. Through the detection of the redox active secondary metabolite, pyocyanin, we demonstrate that a dressing can be produced that will detect the presence of pyocyanin across clinically relevant concentrations. Furthermore, we show that this sensor can be used to identify the presence of pyocyanin in a culture of P. aeruginosa. Overall, the sensor substrate presented in this paper represents the first step toward a new dressing with the capacity to promote wound healing, detect the presence of infection and release antimicrobial drugs, on demand, to optimized healing.

Clinical and Laboratory Associations with Methotrexate Metabolism Gene Polymorphisms in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that causes loss of joint function and significantly reduces quality of life. Plasma metabolite concentrations of disease-modifying anti-rheumatic drugs (DMARDs) can influence treatment efficacy and toxicity. This study explored the relationship between DMARD-metabolising gene variants and plasma metabolite levels in RA patients. DMARD metabolite concentrations were determined by tandem mass-spectrometry in plasma samples from 100 RA patients with actively flaring disease collected at two intervals. Taqman probes were used to discriminate single-nucleotide polymorphism (SNP) genotypes in cohort genomic DNA: rs246240 (ABCC1), rs1476413 (MTHFR), rs2231142 (ABCG2), rs3740065 (ABCC2), rs4149081 (SLCO1B1), rs4846051 (MTHFR), rs10280623 (ABCB1), rs16853826 (ATIC), rs17421511 (MTHFR) and rs717620 (ABCC2). Mean plasma concentrations of methotrexate (MTX) and MTX-7-OH metabolites were higher (p < 0.05) at baseline in rs4149081 GA genotype patients. Patients with rs1476413 SNP TT or CT alleles have significantly higher (p < 0.001) plasma poly-glutamate metabolites at both study time points and correspondingly elevated disease activity scores. Patients with the rs17421511 SNP AA allele reported significantly lower pain scores (p < 0.05) at both study intervals. Genotyping strategies could help prioritise treatments to RA patients most likely to gain clinical benefit whilst minimizing toxicity.

Bacterial Colonization of the Hospitalized Newborn: Competition Between Staphylococcus aureus and Staphylococcus epidermidis.

BACKGROUND: In adults, Staphylococcus epidermidis and Staphylococcus aureus compete for colonization of the nasal mucosa and S. epidermidis strains that produce the Esp serine protease eradicate S. aureus nasal colonization. Whether similar phenomena are seen in newborn infants is unknown. METHODS: Nasal swabs were obtained on admission and discharge from newborn infants (n = 90 and 83, respectively) in the neonatal intensive care unit at UC Davis Children's Hospital. Swabs were cultured for S. aureus and S. epidermidis. S. epidermidis isolates were tested for Esp expression, overall secreted protease activity and biofilm inhibition. RESULTS: No infant had S. aureus on admission. S. epidermidis colonization was rare on admission in inborn infants (2.5%), but common in infants transferred from referring hospitals (50%). At discharge, most infants (96%) were colonized by staphylococci. S. aureus colonization was less common in infants with S. epidermidis colonization (9%) and more common in infants without S. epidermidis (77%) (relative risk of S. aureus colonization in infants colonized with S. epidermidis 0.18, 95% confidence interval: 0.089-0.34, P < 0.0001). Compared with S. epidermidis strains from infants without S. aureus, S. epidermidis from infants co-colonized with S. aureus had lower total proteolytic enzyme activity and decreased biofilm inhibition capacity, but did not have lower frequency of Esp positivity. CONCLUSIONS: In hospitalized neonates, S. epidermidis colonization has a protective effect against S. aureus colonization. Secretion of proteases by S. epidermidis is a possible mechanism of inhibition of S. aureus colonization; however, in this cohort of neonates, the source of major protease activity is likely other than Esp.

A wearable phototherapy device utilizing micro-LEDs.

A conformable device for wearable phototherapy applications is presented. The device consists of a 1 mm thick elastomeric membrane edge-lit by specially fabricated micro-sized LEDs. Nanoparticle based scattering films are utilized to extract light and a uniform emission of 15 µW/cm2 is reported over an area of 2 cm2.

Towards non-invasive characterisation of coronary stent re-endothelialisation - An in-vitro, electrical impedance study.

The permanent implantation of a stent has become the most common method for ameliorating coronary artery narrowing arising from atherosclerosis. Following the procedure, optimal arterial wall healing is characterised by the complete regrowth of an Endothelial Cell monolayer over the exposed stent surface and surrounding tissue, thereby reducing the risk of thrombosis. However, excessive proliferation of Smooth Muscle Cells, within the artery wall can lead to unwanted renarrowing of the vessel lumen. Current imaging techniques are unable to adequately identify re-endothelialisation, and it has previously been reported that the stent itself could be used as an electrode in combination with electrical impedance spectroscopic techniques to monitor the post-stenting recovery phase. The utility of such a device will be determined by its ability to characterise between vascular cell types. Here we present in-vitro impedance spectroscopy measurements of pulmonary artery porcine Endothelial Cells, Human Umbilical Vein Endothelial Cells and coronary artery porcine Smooth Muscle Cells grown to confluence over platinum black electrodes in clinically relevant populations. These measurements were obtained, using a bespoke impedance spectroscopy system that autonomously performed impedance sweeps in the 1kHz to 100kHz frequency range. Analysis of the reactance component of impedance revealed distinct frequency dependent profiles for each cell type with post confluence reactance declines in Endothelial Cell populations that have not been previously reported. Such profiles provide a means of non-invasively characterising between the cell types and give an indication that impedance spectroscopic techniques may enable the non-invasive characterisation of the arterial response to stent placement.

In vitro activity of isavuconazole against fluconazole-resistant isolates of Histoplasma capsulatum.

No clinical trials for histoplasmosis have been performed with the newer azoles, leaving itraconazole as the azole of choice. In vitro studies suggest that Histoplasma capsulatum from patients that relapse on fluconazole has higher minimum inhibitory concentrations (MICs) to fluconazole and voriconazole but not itraconazole and posaconazole. The newest azole, isavuconazole, shares structural similarity to voriconazole, but to date nobody has explored emergence of resistance. In vitro susceptibilities to isavucoanzole and fluconazole were performed on previously obtained isolates from the patients who relapsed on fluconazole therapy. Susceptibilities were determined by NCCLS method M27A developed for yeasts, as modified for H. capsulatum. The change in the MIC from the primary to the relapse isolate was tested using Wilcoxon Rank-Sum for paired data. Among the primary isolates, the median MICs were 1.0 (range 0.25 to 4.0) µg/ml for fluconazole and ≤0.007 (range ≤0.007 to 0.015) µg/ml for isavuconazole. In the group of relapsed isolates, the median MICs rose to 8.0 (range 0.25 to 64.0) µg/ml for fluconazole and remained unchanged at ≤0.007 (range ≤0.007 to 0.015) µg/ml for isavuconazole (P < .001). Only one isolate exhibited a nonsignificant increase in MIC to isavuconazole. Histoplasma isolates from patients who relapsed on fluconazole did not have an elevation in MICs to isavuconazole. This differs from the results previously seen with voriconazole and suggests that despite a closer structural similarity to voriconazole than itraconazole and posaconazole, isavuconazole has a higher barrier to resistance and may be effective as therapy for histoplasmosis.

Peritoneal fluid biomarkers in the detection of colorectal anastomotic leaks: a systematic review.

PURPOSE: Anastomotic leak (AL) in colorectal surgery leads to significant morbidity, mortality and poorer oncological outcomes. Diagnosis of AL is frequently delayed as current methods of detection are not 100% sensitive or specific. 'Biomarkers', such as cytokines and markers of ischaemia, from the milieu of the anastomosis may aid early detection. This paper aims to review the evidence for their role in AL detection, allowing identification of targets for future research. METHODS: A systematic review was performed using PubMed, MEDLINE and Cochrane Library databases. Papers concerning detection or prediction of AL with biomarkers were identified. References within the papers were used to identify further relevant articles. RESULTS: Research has taken place in small cohorts with varying definitions of AL. Lactate has consistently been shown to be elevated in patients with intra-abdominal complications and ALs. pH on post-operative day 3 showed excellent specificity. Despite mixed results, a meta-analysis found that the cytokines tumour necrosis factor-α and interleukin-6 were elevated early in AL. Detection of bacteria in drain fluid by RT-PCR has good specificity but a high rate of false positives. CONCLUSIONS: Peritoneal cytokines, lactate and pH have the potential to identify AL early. The consistency of the results for lactate and pH, alongside the fact that they are easy, quick and inexpensive to test, makes them the most attractive targets. Studies in larger cohorts with standardized definitions of AL are required to clarify their usefulness. Emerging biosensor technology may facilitate the development of small, low-cost and degradable intra-abdominal devices to measure peritoneal fluid biomarkers.

A wearable wound moisture sensor as an indicator for wound dressing change: an observational study of wound moisture and status.

Wound moisture is known to be a key parameter to ensure optimum healing conditions in wound care. This study tests the moisture content of wounds in normal practice in order to observe the moisture condition of the wound at the point of dressing change. This study is also the first large-scale observational study that investigates wound moisture status at dressing change. The WoundSense sensor is a commercially available moisture sensor which sits directly on the wound in order to find the moisture status of the wound without disturbing or removing the dressing. The results show that of the 588 dressing changes recorded, 44·9% were made when the moisture reading was in the optimum moisture zone. Of the 30 patients recruited for this study, 11 patients had an optimum moisture reading for at least 50% of the measurements before dressing change. These results suggest that a large number of unnecessary dressing changes are being made. This is a significant finding of the study as it suggests that the protocols currently followed can be modified to allow fewer dressing changes and less disturbance of the healing wound bed.

Pseudomonas aeruginosa can be detected in a polymicrobial competition model using impedance spectroscopy with a novel biosensor.

Electrochemical Impedance Spectroscopy (EIS) is a powerful technique that can be used to elicit information about an electrode interface. In this article, we highlight six principal processes by which the presence of microorganisms can affect impedance and show how one of these--the production of electroactive metabolites--changes the impedance signature of culture media containing Pseudomonas aeruginosa. EIS, was used in conjunction with a low cost screen printed carbon sensor to detect the presence of P. aeruginosa when grown in isolation or as part of a polymicrobial infection with Staphylococcus aureus. By comparing the electrode to a starting measurement, we were able to identify an impedance signature characteristic of P. aeruginosa. Furthermore, we are able to show that one of the changes in the impedance signature is due to pyocyanin and associated phenazine compounds. The findings of this study indicate that it might be possible to develop a low cost sensor for the detection of P. aeruginosa in important point of care diagnostic applications. In particular, we suggest that a development of the device described here could be used in a polymicrobial clinical sample such as sputum from a CF patient to detect P. aeruginosa.

Development of a highly sensitive and specific blastomycosis antibody enzyme immunoassay using Blastomyces dermatitidis surface protein BAD-1.

Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

Development of wearable sensors for tailored patient wound care.

In recent years a specialist interest has developed worldwide in advanced wound management for difficult to heal chronic wounds. Further progress in advanced wound management will require an improvement in personalized medicine for the patient and in particular an improvement in the availability of diagnostic tests and parameters that fulfil clinical need in wound management decisions. However, without easy to use sensors for nurses and carers these potentially important near-patient diagnostic parameters will not enter clinical diagnostics. This study focuses on a number of metrics for wound condition and wound healing: wound fluid pH, wound moisture, and wound matrix metalloproteinases (MMP) enzyme activity. To observe these important markers state of the art sensors have been developed that are based on inexpensive sensing technologies that can be integrated within wound dressings. These sensors will enable the wound healing markers to be studied and profiled in clinics which will further enhance the understanding of these markers and their relationship in the complex healing process involved in chronic wound healing.

Development of a diagnostic device to detect different Pseudomonas aeruginosa phenotypes in medically relevant contexts.

Pseudomonas aeruginosa, widely present in the environment, is well known for its ability to cause infection in immune compromised individuals. For example, P. aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). Here, we describe how Electrochemical Impedance Spectroscopy (EIS) can be used to detect the presence of four different strains of P. aeruginosa. Using a low cost, screen printed carbon electrode significant changes can be seen in impedance data in the presence of P. aeruginosa after 24 hours. Furthermore, through the use of a normalization technique whereby the phase angle of the impedance (a commonly used parameter) is divided by a starting measurement, it is possible to identify differences between a non-mucoid and mucoid strain of P. aeruginosa. Sensors based upon the techniques described here could be used in a number of healthcare scenarios, where there is a need for low cost, real time detection of P. aeruginosa, such as CF.

Histoplasma capsulatum preferentially induces IDO in the lung.

Indoleamine 2,3 dioxygenase (IDO) plays an important role in immunoregulation as it is involved in downregulating immune responses to infections. We sought to characterize IDO activity in histoplasmosis and to do so, C57Bl6 mice were infected intranasally with Histoplasma capsulatum. After infection, lung and spleen IDO activity was assessed by HPLC and IDO expression by qRT-PCR. The distribution of IDO was determined by immunohistochemical staining. Cytokine levels were measured in lung and spleen homogenates using cytokine bead array. Fungal burden was quantified by culture. Subcutaneous pellets containing methyltryptophane (1-MT) were employed to inhibit IDO in vivo. Histoplasma infection strongly induced functional lung IDO, with activity at its highest at weeks 1 and 2 and then decreased thereafter as the mice cleared the infection. Lung IDO activity positively correlated with the fungal burden (Rho = 0.845), interferon-γ (Rho = 0.839) and tumor necrosis factor-α (Rho = 0.791) levels, P < 0.001. In contrast, spleen IDO activity was not induced despite high infection burden and cytokine levels. IDO expressing cells were predominately located at the ring edge of Histoplasma-induced granulomas. IDO inhibition prior to infection reduced fungal burdens and inflammation in lungs and spleen. Histoplasma preferentially induces lung IDO, as early as one week after infection. IDO appears to modulate the immune response to Histoplasma infection.

Towards minimally invasive monitoring for gastroenterology - An external Squamocolumnar Junction Locator.

Transient lower oesophageal sphincter relaxations (TLOSRs) occur frequently and are the main mechanism of acid reflux. The only means of currently detecting TLOSRs is intra-luminal manometry and the probes themselves may stimulate TLOSRs. The squamo-columnar junction moves 4-5 centimeters proximally during TLOSRs and this provides a means of detecting such episodes. The objective of this work is to develop a sensor system capable of detecting the movement of a miniature magnet attached to the squamo-columnar junction from outside the body and thus allow detection of TLOSRs without the artifact associated with intraluminal detection probes. A GaAs Hall effect sensor was selected and an alternating current supply was developed with a combination of filters and a Phase Sensitive Detector, to detect the magnet. The oscillation frequency of the current was chosen in order to reduce electronic noise, and filtering outside this frequency means the signal to noise ratio was greatly improved. The phase sensitive detector was employed to accurately convert the amplitude of the sensor's output to a DC signal. With the addition of paired Flux Concentrators increases the range up to 10.2 centimetres, an improvement of 580% over commercial Hall effect sensors. The AC circuit and flux concentrator device far exceeds the sensitivity of the current Hall effect sensors supplied in the market, by rejecting noise and providing accurate measurement over significantly larger distances. The development of this sensor has applications beyond this specific medical device.

Blastomyces dermatitidis antigen detection by quantitative enzyme immunoassay.

The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture- or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection.