A Proposed Theoretical Framework for Clinical Judgment in EMS.

In the prehospital setting, EMS clinicians are challenged by the need to assess and treat patients who are clinically undifferentiated with a large constellation of possible medical problems. In addition to possessing a large and diverse set of knowledge, skills, and abilities, EMS clinicians must integrate a plethora of environmental, patient, and event specific cues in their clinical decision-making processes. To date, there is no theoretical framework to capture the complex process that characterizes the prehospital experience from dispatch to handoff, the interface between cues and on-scene information and assessments, while incorporating the importance of leadership and communication. To fill this gap, we propose a theoretical framework for clinical judgment in the prehospital setting that builds upon previously defined methodologies and applies them to the clinical practice of EMS clinicians throughout the EMS experience.

Virulence Evolution of Pathogens That Can Grow in Reservoir Environments.

AbstractMany pathogens reside in environmental reservoirs within which they can reproduce and from which they can infect hosts. These facultative pathogens experience different selective pressures in host-associated environments and reservoir environments. Heterogeneous selective pressures have the potential to influence the virulence evolution of these pathogens. Previous research has examined how environmental transmission influences the selective pressures shaping the virulence of pathogens that cannot reproduce in environmental reservoirs, yet many pathogens of humans, crop plants, and livestock can reproduce in these environments. We build on this work to examine how reproduction in reservoirs influences disease dynamics and virulence evolution in a simple facultative pathogen model. We use adaptive dynamics to examine the evolutionary dynamics of facultative pathogens under potential trade-offs between transmission and virulence, shedding and virulence, and reservoir persistence and virulence. We then perform critical function analysis to generalize the results independent of specific trade-off assumptions. We determine that diverse virulence strategies, sometimes resulting from evolutionary bistability or evolutionary branching conditions, are expected for facultative pathogens. Our findings motivate research establishing which trade-offs most strongly influence the virulence evolution of facultative pathogens.

Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes.

Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulated into 13 µm thick hydrogel layers at a density of 90 cells/mm2, enabling parallel monitoring of 2.8 × 104 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 µm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A. tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.

Identification of Critical Surface Parameters Driving Lectin-Mediated Capture of Bacteria from Solution.

Lectin-functional interfaces are useful for isolation of bacteria from solution because they are low-cost and allow nondestructive, reversible capture. This study provides a systematic investigation of physical and chemical surface parameters that influence bacteria capture over lectin-functionalized polymer interfaces and then applies these findings to construct surfaces with significantly enhanced bacteria capture. The designer block copolymer poly(glycidyl methacrylate)- block-poly(vinyldimethyl azlactone) was used as a lectin attachment layer, and lectin coupling into the polymer film through azlactone-lectin coupling reactions was first characterized. Here, experimental parameters including polymer areal chain density, lectin molecular weight, and lectin coupling buffer were systematically varied to identify parameters driving highest azlactone conversions and corresponding lectin surface densities. To introduce physical nanostructures into the attachment layer, nanopillar arrays (NPAs) of varied heights (300 and 2100 nm) were then used to provide an underlying surface template for the functional polymer layer. Capture of Escherichia coli on lectin-polymer surfaces coated over both flat and NPA surfaces was then investigated. For flat polymer interfaces, bacteria were detected on the surface after incubation at a solution concentration of 103 cfu/mL, and a corresponding detection limit of 1.7 × 103 cfu/mL was quantified. This detection limit was 1 order of magnitude lower than control lectin surfaces functionalized with standard, carbodiimide coupling chemistry. NPA surfaces containing 300 nm tall pillars further improved the detection limit to 2.1 × 102 cfu/mL, but also reduced the viability of captured cells. Finally, to investigate the impact of cell surface parameters on capture, we used Agrobacterium tumefaciens cells genetically modified to allow manipulation of exopolysaccharide adhesin production levels. Statistical analysis of surface capture levels revealed that lectin surface density was the primary factor driving capture, as opposed to exopolysaccharide adhesin expression. These findings emphasize the critical importance of the synthetic interface and the development of surfaces that combine high lectin densities with tailored physical features to drive high levels of capture. These insights will aid in design of biofunctional interfaces with physicochemical surface properties favorable for capture and isolation of bacteria cells from solutions.

Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants.

Many important pathogens maintain significant populations in highly disparate disease and non-disease environments. The consequences of this environmental heterogeneity in shaping the ecological and evolutionary dynamics of these facultative pathogens are incompletely understood. Agrobacterium tumefaciens, the causative agent for crown gall disease of plants has proven a productive model for many aspects of interactions between pathogens and their hosts and with other microbes. In this review, we highlight how this past work provides valuable context for the use of this system to examine how heterogeneity and transitions between disease and non-disease environments influence the ecology and evolution of facultative pathogens. We focus on several features common among facultative pathogens, such as the physiological remodelling required to colonize hosts from environmental reservoirs and the consequences of competition with host and non-host associated microbiota. In addition, we discuss how the life history of facultative pathogens likely often results in ecological tradeoffs associated with performance in disease and non-disease environments. These pathogens may therefore have different competitive dynamics in disease and non-disease environments and are subject to shifting selective pressures that can result in pathoadaptation or the within-host spread of avirulent phenotypes.

Microbial population and community dynamics on plant roots and their feedbacks on plant communities.

The composition of the soil microbial community can be altered dramatically due to association with individual plant species, and these effects on the microbial community can have important feedbacks on plant ecology. Negative plant-soil feedback plays primary roles in maintaining plant community diversity, whereas positive plant-soil feedback may cause community conversion. Host-specific differentiation of the microbial community results from the trade-offs associated with overcoming plant defense and the specific benefits associated with plant rewards. Accumulation of host-specific pathogens likely generates negative feedback on the plant, while changes in the density of microbial mutualists likely generate positive feedback. However, the competitive dynamics among microbes depends on the multidimensional costs of virulence and mutualism, the fine-scale spatial structure within plant roots, and active plant allocation and localized defense. Because of this, incorporating a full view of microbial dynamics is essential to explaining the dynamics of plant-soil feedbacks and therefore plant community ecology.

Differences in Paramedic Fatigue before and after Changing from a 24-hour to an 8-hour Shift Schedule: A Case Report.

Emergency medical services (EMS) clinicians often work 24-hour shifts. There is a growing body of literature, with an elevated level of concern among EMS leaders that longer shifts contribute to fatigued workers and negative safety outcomes. However, many questions remain about shift length, fatigue, and outcomes. We describe a case of a 26-year-old male paramedic who switched shift schedules during the midpoint of a randomized trial that addressed fatigue in EMS workers (clinicaltrials.gov identifier: NCT02063737). The participant (case) began the study working full-time with a critical care, advanced life support EMS system that utilized 24-hour shifts. He then transitioned to an EMS system that deploys workers on 8-hour shifts. Per protocol for the randomized trial, the participant completed a battery of sleep health and fatigue surveys at baseline and at the end of 90 days of study. He also reported perceived fatigue, sleepiness, and difficulty with concentration at the beginning, every 4 hours during, and at the end of scheduled shifts, for a total of ten 24-hour shifts and twenty-four 8-hour shifts. We discuss differences in measures taken before and after switching shift schedules, and highlight differences in fatigue, sleepiness, and difficulty with concentration taken at the end of all 34 scheduled shifts stratified by shift duration (24 hours versus 8 hours). Findings from this case report present a unique opportunity to 1) observe and analyze a phenomenon that has not been investigated in great detail in the EMS setting; and 2) address an issue of significance to employers and EMS clinicians alike.

Adding urine and saliva toxicology to SBIRT for drug screening of new patients.

BACKGROUND AND OBJECTIVES: To determine illicit drug use among new patients in primary medical care who denied using "street drugs" during Screening, Brief Intervention and Referral to Treatment (SBIRT). METHODS: 96 new patients who denied use of "street drugs" were tested for drugs as part of routine SBIRT screening. RESULTS: Of those tested, 14.6% of those with urine specimens and 4.1% of those with saliva specimens tested positive for illicit drugs. DISCUSSION AND CONCLUSIONS: Drug toxicology can detect unreported illicit drug use during SBIRT screening, with urine being superior to saliva. SCIENTIFIC SIGNIFICANCE: Drug toxicology can increase the effectiveness of SBIRT screening in primary care medical clinics.

Transgenic models of Alzheimer's disease: better utilization of existing models through viral transgenesis.

Animal models have been used for decades in the Alzheimer's disease (AD) research field and have been crucial for the advancement of our understanding of the disease. Most models are based on familial AD mutations of genes involved in the amyloidogenic process, such as the amyloid precursor protein (APP) and presenilin 1 (PS1). Some models also incorporate mutations in tau (MAPT) known to cause frontotemporal dementia, a neurodegenerative disease that shares some elements of neuropathology with AD. While these models are complex, they fail to display pathology that perfectly recapitulates that of the human disease. Unfortunately, this level of pre-existing complexity creates a barrier to the further modification and improvement of these models. However, as the efficacy and safety of viral vectors improves, their use as an alternative to germline genetic modification is becoming a widely used research tool. In this review we discuss how this approach can be used to better utilize common mouse models in AD research. This article is part of a Special Issue entitled: Animal Models of Disease.

Leptin regulates amyloid ß production via the γ-secretase complex.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, affecting an estimated 5.3million people in the United States. While many factors likely contribute to AD progression, it is widely accepted that AD is driven by the accumulation of ß-amyloid (Aß), a small, fibrillogenic peptide generated by the sequential proteolysis of the amyloid precursor protein by the ß- and γ-secretases. Though the underlying causes of Aß accumulation in sporadic AD are myriad, it is clear that lifestyle and overall health play a significant role. The adipocyte-derived hormone leptin has varied systemic affects, including neuropeptide release and neuroprotection. A recent study by Lieb et al. (2009) showed that individuals with low plasma leptin levels are at greater risk of developing AD, through unknown mechanisms. In this report, we show that plasma leptin is a strong negative predictor of Aß levels in the mouse brain, supporting a protective role for the hormone in AD onset. We also show that the inhibition of Aß accumulation is due to the downregulation of transcription of the γ-secretase components. On the other hand, ß-secretase expression is either unchanged (BACE1) or increased (BACE2). Finally, we show that only presenilin 1 (PS1) is negatively correlated with plasma leptin at the protein level (p<0.0001). These data are intriguing and may highlight a role for leptin in regulating the onset of amyloid pathology and AD.

Non-additive costs and interactions alter the competitive dynamics of co-occurring ecologically distinct plasmids.

Plasmids play an important role in shaping bacterial evolution and adaptation to heterogeneous environments. As modular genetic elements that are often conjugative, the selective pressures that act on plasmid-borne genes are distinct from those that act on the chromosome. Many bacteria are co-infected by multiple plasmids that impart niche-specific phenotypes. Thus, in addition to host-plasmid dynamics, interactions between co-infecting plasmids are likely to be important drivers of plasmid population dynamics, evolution and ecology. Agrobacterium tumefaciens is a facultative plant pathogen that commonly harbours two distinct megaplasmids. Virulence depends on the presence of the tumour-inducing (Ti) plasmid, with benefits that are primarily restricted to the disease environment. Here, we demonstrate that a second megaplasmid, the At plasmid, confers a competitive advantage in the rhizosphere. To assess the individual and interactive costs of these plasmids, we generated four isogenic derivatives: plasmidless, pAt only, pTi only and pAtpTi, and performed pairwise competitions under carbon-limiting conditions. These studies reveal a low cost to the virulence plasmid when outside of the disease environment, and a strikingly high cost to the At plasmid. In addition, the costs of pAt and pTi in the same host were significantly lower than predicted based on single plasmid costs, signifying the first demonstration of non-additivity between naturally occurring co-resident plasmids. Based on these empirically demonstrated costs and benefits, we developed a resource-consumer model to generate predictions about the frequencies of these genotypes in relevant environments, showing that non-additivity between co-residing plasmids allows for their stable coexistence across environments.

Bleeding Reversal With Antifibrinolytics or Cryoprecipitate Following Thrombolysis for Acute Ischemic Stroke: A Case Series.

Patients who develop an intracerebral hemorrhage (ICH) following thrombolysis in acute ischemic stroke (AIS) have a mortality rate as high as 50%. Treatment options include blood products, such as cryoprecipitate, or antifibrinolytics, such as tranexamic acid (TXA) or ε-aminocaproic acid (EACA). Current guidelines recommend cryoprecipitate first-line despite limited data to support one agent over another. In addition, compared to antifibrinolytics, cryoprecipitate is higher in cost and requires thawing before use. This case series seeks to characterize the management of thrombolytic reversal at a single institution as well as provide additional evidence for antifibrinolytics in this setting. Patients were included for a retrospective review if they met the following criteria: presented between January 2011-January 2017, were >18 years of age, were admitted for AIS, received a thrombolytic, and received TXA EACA, or cryoprecipitate. Twelve patients met the inclusion criteria. Ten (83.3%) developed an ICH, one (8.3%) experienced gastrointestinal bleeding, and one (8.3%) had bleeding at the site of knee arthroscopy. Eleven patients received cryoprecipitate (median dose: 10 units), three received TXA (median dose: 1,000 mg), and one patient received EACA (13 g). TXA was administered faster than the first blood product at a mean time of 19 min and 137 min, respectively. Hemorrhagic expansion (N = 8, 66.67%) and inhospital mortality (N = 7, 58.3%) were high. While limited by its small sample size, this case series demonstrates significant variability in reversal strategies for thrombolysis-associated bleeding. It also provides additional evidence for the role of antifibrinolytics in this setting.

Assessment of long-term medication adherence with cystic fibrosis: An integrated approach.

INTRODUCTION: Cystic fibrosis (CF) is a genetic disorder that creates a multisystem pathology resulting in complex treatment regimens. In 2014, 43% of people with CF at an academic medical center experienced medication acquisition barriers. The creation of an integrated specialty pharmacy with an embedded CF team pharmacist was launched in 2016. In addition to filling specialty medications, this specialty pharmacy filled all patient medications through a service called total care pharmacy (TCP). This service was hypothesized to positively impact medication adherence. METHODS: Adherence analysis was performed by utilizing the proportion of days covered (PDC). PDC was analyzed during years 1, 2, and 3 of therapy. PDC was calculated for medications with at least three fills during each year. Patients with PDC less than 80% were considered nonadherent and underwent manual chart review to identify a documented reason for nonadherence. RESULTS: Patients in the first year of dornase alfa therapy had significantly higher adherence in the TCP cohort compared to non-TCP (81.3% PDC vs. 66.0%; p = .006), which was largely driven by adult patients (73.3% vs. 56.5% for pediatric). Analysis of other medications and groups did not yield statistically significant differences. Many patients who had been classified as nonadherent had valid clinical reasons that explained gaps in therapy. CONCLUSIONS: When filling medications at a specialty pharmacy integrated within the academic medication center, dornase alfa adherence was higher in the TCP group. Further studies comparing TCP with services offered by pharmacies external to the health system would better characterize the impact of TCP services.

Photo-addressable microwell devices for rapid functional screening and isolation of pathogen inhibitors from bacterial strain libraries.

Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen Agrobacterium tumefaciens sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens.

Screening for quality with process analytical technology in a health-system pharmacy: A primer.

PURPOSE: The University of Kentucky Drug Quality Study team briefly reviews the growing concerns over pharmaceutical manufacturing quality in the globalized environment, reviews the historical approach by the US Food and Drug Administration (FDA) that prioritizes process over product in enforcing quality with manufacturers, reviews the science of process analytical technology (PAT) such as near-infrared (NIR) spectroscopy, illustrates the use of PAT methods for assessing uniformity and quality in injectable pharmaceuticals, and demonstrates the application of NIR spectroscopy in a health-system pharmacy setting while maintaining current good practice quality guidelines and regulations (cGxP). SUMMARY: Given that the current approach to monitoring quality in pharmaceutical manufacturing was developed in the late 1960s at a time when manufacturing was mostly domestic, the current approach prioritizes process over product, and the global footprint of manufacturing is straining federal resources to fulfill their task of monitoring quality, an approach to augment the quality monitoring process has been developed. PAT methodologies are supported by FDA for monitoring quality and offer a fast, low-cost, nondestructive solution. Given that the Accreditation Council for Pharmacy Education has not required qualitative/quantitative analysis and drug assaying in the pharmacy curriculum for several decades, the authors spend time explaining the science behind one of these PAT methodologies, NIR spectroscopy. This primer reviews the application of this technology in the health-system pharmacy setting and the relevant clinical applications. CONCLUSION: Utilizing PAT methodologies such as NIR spectroscopy, health-system pharmacies can gain insights about whether process controls are in place or lacking in FDA-approved formulations.

Application of Near-Infrared Spectroscopy for Screening of Chlorothiazide Sodium Vials.

Chlorothiazide sodium for injection, USP, is a diuretic and antihypertensive medication in the form of a white or practically white, sterile, lyophilized powder. Each vial contains 500 mg of chlorothiazide sodium, equivalent to 500 mg of chlorothiazide, and 250 mg of mannitol as an inactive ingredient. The pH is adjusted with sodium hydroxide. Chlorothiazide sodium has a molecular weight of 317.71 amu. Since 2020 there have been multiple national shortages of chlorothiazide. Recent studies target chlorothiazide's low bioavailability, aiming to enhance it through nanoparticle production via a supercritical method. The drug's solubility in supercritical carbon dioxide (scCO2) is vital, with measurements ranging from 0.417×10-5 to 1.012×10-5 mole fraction under specific conditions. Adding co-solvents, like ethanol, DMSO, and acetone, to scCO2 boosts solubility, with ethanol proving most effective, enhancing solubility by 2.02-11.75 times. Intra-lot variability was discovered in a sample of a lot of chlorothiazide sodium by the University of Kentucky Drug Quality Task Force. Two vials of six screened in one lot were displaced from the center of the lot by 4.0 and 4.2 SDs, respectively. Inter-lot variability was confirmed in the near-IR spectra of 204 vials obtained from 28 different lots of chlorothiazide sodium. Using full spectrum BEST analysis 13 vials (6.4%) were outliers.

Spectrometric Analysis of Dantrolene Sodium.

Dantrolene sodium is a direct-acting skeletal muscle relaxant. Dantrolene sodium for injection is indicated, along with suitable supportive measures, for the management of sudden, severe hypermetabolism of skeletal muscle typical of malignant hyperthermia crises in patients of any age. The formulation scanned in this work was designed to be injected intravenously. Intra-lot and inter-lot variability in the spectra of REVONTO™ (dantrolene sodium) was measured in the Drug Quality Study (DQS) using Fourier transform near-infrared spectrometry (FTNIR). Spectra of 69 vials from lot 20REV01A contained two groups (n1=56 vials, n2=13 vials) when scanned with an FTNIR. The two groups of spectra in lot 20REV01A were found to be 66.7 SDs apart using a subcluster detection test, suggesting that the two groups were manufactured differently. As a result, all available samples of dantrolene were examined. A library of spectra of 141 vials of dantrolene from 4 lots were found to contain 3 separate groups, also suggesting that different vials contain different materials.

Quality Variations in Thyrotropin Alfa.

Thyrotropin alfa is a heterodimeric glycoprotein containing human thyroid stimulating hormone (TSH). It is used as an adjunctive diagnostic tool for serum thyroglobulin (Tg) testing with or without radioiodine imaging in the follow-up of patients with well-differentiated thyroid cancer who have previously undergone thyroidectomy. Inter-lot variability in the Fourier transform near-infrared spectra of 30 samples obtained from four separate lots of Thyrogen® was detected in the Drug Quality Study (DQS). The vials fell into two distinct groups (rtst = 0.90, rlim= 0.98, p=0.02). In addition, one vial of the 30 (3%) appeared 4.7 multidimensional SDs from all of the other vials, suggesting that it also represents a different material.