Intestinal Bacteroides modulates inflammation, systemic cytokines, and microbial ecology via propionate in a mouse model of cystic fibrosis.

Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1ß-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.

Development of the intestinal microbiome in cystic fibrosis in early life.

Cystic fibrosis (CF) is a heritable disease that causes altered physiology at mucosal sites; these changes result in chronic infections in the lung, significant gastrointestinal complications as well as dysbiosis of the gut microbiome, although the latter has been less well explored. Here, we describe the longitudinal development of the gut microbiome in a cohort of children with CF (cwCF) from birth through early childhood (0-4 years of age) using 16S rRNA gene amplicon sequencing of stool samples as a surrogate for the gut microbiota. Similar to healthy populations, alpha diversity of the gut microbiome increases significantly with age, but diversity plateaus at ~2 years of age for this CF cohort. Several taxa that have been associated with dysbiosis in CF change with age toward a more healthy-like composition; notable exceptions include Akkermansia, which decreases with age, and Blautia, which increases with age. We also examined the relative abundance and prevalence of nine taxa associated with CF lung disease, several of which persist across early life, highlighting the possibility of the lung being seeded directly from the gut early in life. Finally, we applied the Crohn's Dysbiosis Index to each sample, and found that high Crohn's-associated dysbiosis early in life (<2 years) was associated with significantly lower Bacteroides in samples collected from 2 to 4 years of age. Together, these data comprise an observational study that describes the longitudinal development of the CF-associated gut microbiota and suggest that early markers associated with inflammatory bowel disease may shape the later gut microbiota of cwCF. IMPORTANCE Cystic fibrosis is a heritable disease that disrupts ion transport at mucosal surfaces, causing a buildup of mucus and dysregulation of microbial communities in both the lungs and the intestines. Persons with CF are known to have dysbiotic gut microbial communities, but the development of these communities over time beginning at birth has not been thoroughly studied. Here, we describe an observation study following the development of the gut microbiome of cwCF throughout the first 4 years of life, during the critical window of both gut microbiome and immune development. Our findings indicate the possibility of the gut microbiota as a reservoir of airway pathogens and a surprisingly early indication of a microbiota associated with inflammatory bowel disease.

Persistent delay in maturation of the developing gut microbiota in infants with cystic fibrosis.

The healthy human infant gut microbiome undergoes stereotypical changes in taxonomic composition between birth and maturation to an adult-like stable state. During this time, extensive communication between microbiota and the host immune system contributes to health status later in life. Although there are many reported associations between microbiota compositional alterations and disease in adults, less is known about how microbiome development is altered in pediatric diseases. One pediatric disease linked to altered gut microbiota composition is cystic fibrosis (CF), a multi-organ genetic disease involving impaired chloride secretion across epithelia and heightened inflammation both in the gut and at other body sites. Here, we use shotgun metagenomics to profile the strain-level composition and developmental dynamics of the infant fecal microbiota from several CF and non-CF longitudinal cohorts spanning from birth to greater than 36 months of life. We identify a set of keystone species whose prevalence and abundance reproducibly define microbiota development in early life in non-CF infants, but are missing or decreased in relative abundance in infants with CF. The consequences of these CF-specific differences in gut microbiota composition and dynamics are a delayed pattern of microbiota maturation, persistent entrenchment in a transitional developmental phase, and subsequent failure to attain an adult-like stable microbiota. We also detect the increased relative abundance of oral-derived bacteria and higher levels of fungi in CF, features that are associated with decreased gut bacterial density in inflammatory bowel diseases. Our results define key differences in the gut microbiota during ontogeny in CF and suggest the potential for directed therapies to overcome developmental delays in microbiota maturation.

Physician experience with the Epic electronic health record (EHR) system: longitudinal findings from an emergency department implementation.

OBJECTIVES: In June 2019, The Ottawa Hospital launched the Epic electronic health record system, which transitioned all departments from a primarily paper-based system to an electronic system using a 1-day "big bang" approach. We sought to evaluate emergency physicians' satisfaction with system implementation and perception of its impact on clinical practice in an academic emergency department (ED) setting. METHODS: Four electronic surveys were distributed to staff during pre-implementation (1-month prior [May 2019]) and post-implementation (1-month [July 2019], 9-month [March 2020], and 20-month [February 2021]) time periods. 5-point Likert scales were used to rate agreement with statements. Responses were compared using the Cochran-Mantel-Haenszel trend test to assess for significant differences. RESULTS: Response rates were consistent, ranging between 41 and 51%, with the exception of +9 months which was 27%. The majority of respondents were staff, working 8-15 shifts/month, with ≤ 10 years in practice. General satisfaction and confidence improved substantially from pre-implementation to 20 months post-implementation. Personalization sessions were perceived as not effective and lacking in quality, particularly immediately after Epic launch. Although clinical workflow tasks got easier, there were sustained challenges in efficiency and patient flow, including number of patients seen/hour, time spent after shift-end, and time spent on post-shift documentation. CONCLUSIONS: Although satisfaction and system confidence improved over time, there were sustained difficulties in overall efficiency long after implementation, with opportunities for future optimization. Training was lacking in terms of relevance to emergency physician workflow. These factors should be considered in future electronic health record implementations in ED settings.

RéSUMé: OBJECTIFS: En juin 2019, l'Hôpital d'Ottawa a lancé le système de dossiers de santé électroniques Epic, ce qui a permis de faire passer tous les services d'un système principalement basé sur le papier à un système électronique en utilisant une approche " big bang " d'une journée. Nous avons cherché à évaluer la satisfaction des médecins urgentistes quant à la mise en œuvre du système et la perception de son impact sur la pratique clinique dans un service d'urgence universitaire. MéTHODES: Quatre sondages électroniques ont été distribués au personnel pendant les périodes précédant la mise en œuvre (1 mois avant [mai 2019]) et suivant la mise en œuvre (1 mois [juillet 2019], 9 mois [mars 2020] et 20 mois [février 2021]). Des échelles de Likert à 5 points ont été utilisées pour évaluer l'accord avec les énoncés. Les réponses ont été comparées à l'aide du test de tendance de Cochran-Mantel-Haenszel pour évaluer les différences significatives. RéSULTATS: Les taux de réponse étaient constants, allant de 41 % à 51 %, à l'exception de celui de +9 mois qui était de 27 %. La majorité des répondants étaient des employés, travaillant de 8 à 15 quarts par mois, avec moins de 10 ans de pratique. La satisfaction générale et la confiance se sont considérablement améliorées entre la période précédant la mise en œuvre et la période de 20 mois suivant la mise en œuvre. Les sessions de personnalisation ont été perçues comme inefficaces et manquant de qualité, en particulier immédiatement après le lancement d'Epic. Bien que les tâches du flux de travail clinique aient été facilitées, l'efficacité et le flux de patients ont continué à poser problème, notamment le nombre de patients vus par heure, le temps passé après la fin du quart de travail et le temps consacré à la documentation après le quart de travail. CONCLUSIONS: Bien que la satisfaction et la confiance dans le système se soient améliorées au fil du temps, il y a eu des difficultés persistantes dans l'efficacité globale longtemps après la mise en œuvre, avec des possibilités d'optimisation future. La formation manquait de pertinence par rapport au flux de travail des médecins urgentistes. Ces facteurs devraient être pris en compte dans les futures mises en œuvre de dossiers médicaux électroniques dans les services d'urgence.

Temporary In Situ Hydrogel Dressings for Colon Polypectomies.

Currently, no dressings are utilized after removal of polyps during a colonoscopy rendering these tissue sites susceptible to bleeding, sepsis, and perfusion. We report the design specifications, synthesis, and ex vivo evaluation of in situ polymerized hydrogels as colon wound dressings post polypectomy. The hydrogels exhibited varied properties to include moduli between 100 and 16 000 Pa, dissolution times between 4 h to 7 days or longer, swelling up to 200%, and adhesion to colon tissue from 0.1 to 0.4 N/cm2. The hydrogels displayed minimal cytotoxicity, prevented the migration/spread of bacteria, and exhibited rapid gelation, a requirement for application to the lumen of the colon via an endoscope. This work highlights the structure-property relationship of hydrogels prepared from N-hydroxysuccinimide functionalized PEG cross-linkers and hyperbranched polyethylenimines or 4-arm PEG-NH2 star polymers, and their potential as colon wound dressings.

The Gut-Lung Axis in Cystic Fibrosis.

Cystic fibrosis (CF) is a heritable, multiorgan disease that impacts all tissues that normally express cystic fibrosis transmembrane conductance regulator (CFTR) protein. While the importance of the airway microbiota has long been recognized, the intestinal microbiota has only recently been recognized as an important player in both intestinal and lung health outcomes for persons with CF (pwCF). Here, we summarize current literature related to the gut-lung axis in CF, with a particular focus on three key ideas: (i) mechanisms through which microbes influence the gut-lung axis, (ii) drivers of microbiota alterations, and (iii) the potential for intestinal microbiota remediation.

Intubator Performance and Contamination with the Use of Barrier Enclosure Devices: Results from a Simulated COVID-19 Resuscitation.

INTRODUCTION: Medical institutions are using barrier enclosure devices during intubation procedures and other aerosol-generating medical procedures without evidence of their effectiveness or usability, potentially compromising patient care, and provider safety. Our objective was to determine the degree of protection offered by these devices and explore other usability factors for two popular barrier systems. METHODS: A simulated trial comparing an intubation box, a frame and plastic tarp system, and unprotected intubation was performed in an academic emergency department. Ten emergency physicians were recruited to participate. Our primary outcome was the degree of contamination from secretions measured by average surface area exposed to phosphorescent material. Secondary outcomes included: laryngoscopy time and time to barrier application, unsuccessful intubation attempts, and usability ratings for each system. Descriptive statistics were reported for all variables of interest and a linear mixed model was used to analyze contamination and laryngoscopy time. Usability was captured through electronic questionnaires using a five-point Likert scale. RESULTS: Contamination was more prevalent with the box, compared to the frame and tarp, and no device, however, this did not achieve statistical significance (13.2% versus 8.1% versus 12.2%, P = 0.17). A barrier system delayed intubation when compared to using no system (no system = 24.4 s [95% CI 17.3-27.5], frame = 54.4 s [95% CI 13.8-95.0], box = 33.8 s [95% CI 21.4-46.1], P = 0.02). In assessing usability, 30% of users preferred the use of a box barrier, 40% of users preferred the frame, and 30% would not use either in future intubation. CONCLUSIONS: Compared to no barrier protection, an intubation box enclosure offers limited additional protection. A frame and tarp system reduces exposure at the expense of visibility and operator comfort. Finally, barrier systems do not appear to have a clinically significant impact on airway management.

The impact of adoption of an electronic health record on emergency physician work: A time motion study.

OBJECTIVE: We assessed the impact of the transition from a primarily paper-based electronic health record (EHR) to a comprehensive EHR on emergency physician work tasks and efficiency in an academic emergency department (ED). METHODS: We conducted a time motion study of emergency physicians on shift in our ED. Fifteen emergency physicians were directly observed for two 4-hour sessions prior to EHR implementation, during go live, and then during post-implementation. Observers performed continuous observation and measured times for the following tasks: chart review, direct patient care, documentation, physical movement, communication, teaching, handover, and other. We compared time spent on tasks during the 3 phases of transition and analyzed mean times for the tasks per patient and per shift using 2-tailed t test for comparison. RESULTS: Physicians saw fewer patients per shift during go-live (0.51 patient/hour, P < 0.01), patient efficiency increased in post-implementation but did not recover to baseline (-0.31 patient/hour, P = 0.03). From pre-implementation to post-implementation, we observed a trend towards increased physician time spent charting (+54 seconds/patient, P = 0.05) and documenting (+36 seconds/patient, P = 0.36); time spent doing direct patient care trended towards decreasing (-0.43 seconds/patient, P = 0.23). A small percentage of shifts were spent receiving technical support and time spent on teaching activities remained relatively stable during EHR transition. CONCLUSION: A new EHR impacts emergency physician task allocation and several changes are sustained post-implementation. Physician efficiency decreased and did not recover to baseline. Understanding workflow changes during transition to EHR in the ED is necessary to develop strategies to maintain quality of care.

Barrier enclosure use during aerosol-generating medical procedures: A scoping review.

INTRODUCTION: Barrier enclosure devices were introduced to protect against infectious disease transmission during aerosol generating medical procedures (AGMP). Recent discussion in the medical community has led to new designs and adoption despite limited evidence. A scoping review was conducted to characterize devices being used and their performance. METHODS: We conducted a scoping review of formal databases (MEDLINE, Embase, Cochrane Database of Systematic Reviews, CENTRAL, Scopus), grey literature, and hand-searched relevant journals. Forward and reverse citation searching was completed on included articles. Article/full-text screening and data extraction was performed by two independent reviewers. Studies were categorized by publication type, device category, intended medical use, and outcomes (efficacy - ability to contain particles; efficiency - time to complete AGMP; and usability - user experience). RESULTS: Searches identified 6489 studies and 123 met criteria for inclusion (k = 0.81 title/abstract, k = 0.77 full-text). Most articles were published in 2020 (98%, n = 120) as letters/commentaries (58%, n = 71). Box systems represented 42% (n = 52) of systems described, while plastic sheet systems accounted for 54% (n = 66). The majority were used for airway management (67%, n = 83). Only half of articles described outcome measures (54%, n = 67); 82% (n = 55) reporting efficacy, 39% (n = 26) on usability, and 15% (n = 10) on efficiency. Efficacy of devices in containing aerosols was limited and frequently dependent on use of suction devices. CONCLUSIONS: While use of various barrier enclosure devices has become widespread during this pandemic, objective data of efficacy, efficiency, and usability is limited. Further controlled studies are required before adoption into routine clinical practice.

A system uptake analysis and GUIDES checklist evaluation of the Electronic Asthma Management System: A point-of-care computerized clinical decision support system.

OBJECTIVE: Computerized clinical decision support systems (CCDSSs) promise improvements in care quality; however, uptake is often suboptimal. We sought to characterize system use, its predictors, and user feedback for the Electronic Asthma Management System (eAMS)-an electronic medical record system-integrated, point-of-care CCDSS for asthma-and applied the GUIDES checklist as a framework to identify areas for improvement. MATERIALS AND METHODS: The eAMS was tested in a 1-year prospective cohort study across 3 Ontario primary care sites. We recorded system usage by clinicians and patient characteristics through system logs and chart reviews. We created multivariable models to identify predictors of (1) CCDSS opening and (2) creation of a self-management asthma action plan (AAP) (final CCDSS step). Electronic questionnaires captured user feedback. RESULTS: Over 1 year, 490 asthma patients saw 121 clinicians. The CCDSS was opened in 205 of 1033 (19.8%) visits and an AAP created in 121 of 1033 (11.7%) visits. Multivariable predictors of opening the CCDSS and producing an AAP included clinic site, having physician-diagnosed asthma, and presenting with an asthma- or respiratory-related complaint. The system usability scale score was 66.3 ± 16.5 (maximum 100). Reported usage barriers included time and system accessibility. DISCUSSION: The eAMS was used in a minority of asthma patient visits. Varying workflows and cultures across clinics, physician beliefs regarding asthma diagnosis, and relevance of the clinical complaint influenced uptake. CONCLUSIONS: Considering our findings in the context of the GUIDES checklist helped to identify improvements to drive uptake and provides lessons relevant to CCDSS design across diseases.

Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureusin vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner.IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

When personal health data is no longer "personal".

Enacted in 2000, the Canadian Personal Health Information Protection and Electronics Documents Act is an important piece of legislation aimed at safeguarding an individual's right to control their personal health information. Since this time, the world of data and analytics has shifted in terms of our potential to collect, integrate, and analyze both structured and unstructured data. The implications for these data advancements are endless for our healthcare system; however, challenges influenced by our approach to collecting, accessing, and analyzing data as well as patient consent to share personal health information mean public entities lag behind commercial players in harnessing these potential benefits. While there are examples of data analytics application successes, Canadian healthcare continues to lag behind other countries and commercial sectors. We are at a pivot point for system improvements requiring a collective approach to collection, storage, linkage, and application of personal healthcare data. In the chasm of this rests how we address patient consent. All health leaders can play a central role in advancing our application of data for system improvements. Strategies to support health leaders in achieving this potential are outlined in this article.

Altered Stool Microbiota of Infants with Cystic Fibrosis Shows a Reduction in Genera Associated with Immune Programming from Birth.

Previous work from our group indicated an association between the gastrointestinal microbiota of infants with cystic fibrosis (CF) and airway disease in this population. Here we report that stool microbiota of infants with CF demonstrates an altered but largely unchanging within-individual bacterial diversity (alpha diversity) over the first year of life, in contrast to the infants without CF (control cohort), which showed the expected increase in alpha diversity over the first year. The beta diversity, or between-sample diversity, of these two cohorts was significantly different over the first year of life and was statistically significantly associated with airway exacerbations, confirming our earlier findings. Compared with control infants, infants with CF had reduced levels of Bacteroides, a bacterial genus associated with immune modulation, as early as 6 weeks of life, and this significant reduction of Bacteroides spp. in the cohort with CF persisted over the entire first year of life. Only two other genera were significantly different across the first year of life: Roseburia was significantly reduced and Veillonella was significantly increased. Other genera showed differences between the two cohorts but only at selected time points. In vitro studies demonstrated that exposure of the apical face of polarized intestinal cell lines to Bacteroides species supernatants significantly reduced production of interleukin 8 (IL-8), suggesting a mechanism whereby changes in the intestinal microbiota could impact inflammation in CF. This work further establishes an association between gastrointestinal microbiota, inflammation, and airway disease in infants with CF and presents a potential opportunity for therapeutic interventions beginning in early life.IMPORTANCE There is growing evidence for a link between gastrointestinal bacterial communities and airway disease progression in CF. We demonstrate that infants with CF ≤1 year of age show a distinct stool microbiota versus that of control infants of a comparable age. We detected associations between the gut microbiome and airway exacerbation events in the cohort of infants with CF, and in vitro studies provided one possible mechanism for this observation. These data clarify that current therapeutics do not establish in infants with CF a gastrointestinal microbiota like that in healthy infants, and we suggest that interventions that direct the gastrointestinal microbiota closer to a healthy state may provide systemic benefits to these patients during a critical window of immune programming that might have implications for lifelong health.

The Electronic Asthma Management System (eAMS) improves primary care asthma management.

A high prevalence of suboptimal asthma control is attributable to known evidence-practice gaps. We developed a computerised clinical decision support system (the Electronic Asthma Management System (eAMS)) to address major care gaps and sought to measure its impact on care in adults with asthma.This was a 2-year interrupted time-series study of usual care (year 1) versus eAMS (year 2) at three Canadian primary care sites. We included asthma patients aged ≥16 years receiving an asthma medication within the last 12 months. The eAMS consisted of a touch tablet patient questionnaire completed in the waiting room, with real-time data processing producing electronic medical record-integrated clinician decision support.Action plan delivery (primary outcome) improved from zero out of 412 (0%) to 79 out of 443 (17.8%) eligible patients (absolute increase 0.18 (95% CI 0.14-0.22)). Time-series analysis indicated a 30.5% increase in physician visits with action plan delivery with the intervention (p<0.0001). Assessment of asthma control level increased from 173 out of 3497 (4.9%) to 849 out of 3062 (27.7%) eligible visits (adjusted OR 8.62 (95% CI 5.14-12.45)). Clinicians escalated controller therapy in 108 out of 3422 (3.2%) baseline visits versus 126 out of 3240 (3.9%) intervention visits (p=0.12). At baseline, a short-acting ß-agonist alone was added in 62 visits and a controller added in 54 visits; with the intervention, this occurred in 33 and 229 visits, respectively (p<0.001).The eAMS improved asthma quality of care in real-world primary care settings. Strategies to further increase clinician uptake and a randomised controlled trial to assess impact on patient outcomes are now required.

Large care gaps in primary care management of asthma: a longitudinal practice audit.

OBJECTIVES: Care gaps in asthma may be highly prevalent but are poorly characterised. We sought to prospectively measure adherence to key evidence-based adult asthma practices in primary care, and predictors of these behaviours. DESIGN: One-year prospective cohort study employing an electronic chart audit. SETTING: Three family health teams (two academic, one community-based) in Ontario, Canada. PARTICIPANTS: 884 patients (72.1% female; 46.0±17.5 years old) (4199 total visits; 4.8±4.8 visits/patient) assigned to 23 physicians (65% female; practising for 10.0±8.6 years). MAIN OUTCOME MEASURES: The primary outcome was the proportion of visits during which practitioners assessed asthma control according to symptom-based criteria. Secondary outcomes included the proportion of: patients who had asthma control assessed at least once; visits during which a controller medication was initiated or escalated; and patients who received a written asthma action plan. Behavioural predictors were established a priori and tested in a multivariable model. RESULTS: Primary outcome: Providers assessed asthma control in 4.9% of visits and 15.4% of patients. Factors influencing assessment included clinic site (p=0.019) and presenting symptom, with providers assessing control more often during visits for asthma symptoms (35.0%) or any respiratory symptoms (18.8%) relative to other visits (1.6%) (p<0.01). SECONDARY OUTCOMES: Providers escalated controller therapy in 3.3% of visits and 15.4% of patients. Factors influencing escalation included clinic site, presenting symptom and prior objective asthma diagnosis. Escalation occurred more frequently during visits for asthma symptoms (21.0%) or any respiratory symptoms (11.9%) relative to other visits (1.5%) (p<0.01) and in patients without a prior objective asthma diagnosis (3.5%) relative to those with (1.3%) (p=0.025). No asthma action plans were delivered. CONCLUSIONS: Major gaps in evidence-based asthma practice exist in primary care. Targeted knowledge translation interventions are required to address these gaps, and can be tailored by leveraging the identified behavioural predictors. TRIAL REGISTRATION NUMBER: NCT01070095; Pre-results.

Unsupervised Extraction of Stable Expression Signatures from Public Compendia with an Ensemble of Neural Networks.

Cross-experiment comparisons in public data compendia are challenged by unmatched conditions and technical noise. The ADAGE method, which performs unsupervised integration with denoising autoencoder neural networks, can identify biological patterns, but because ADAGE models, like many neural networks, are over-parameterized, different ADAGE models perform equally well. To enhance model robustness and better build signatures consistent with biological pathways, we developed an ensemble ADAGE (eADAGE) that integrated stable signatures across models. We applied eADAGE to a compendium of Pseudomonas aeruginosa gene expression profiling experiments performed in 78 media. eADAGE revealed a phosphate starvation response controlled by PhoB in media with moderate phosphate and predicted that a second stimulus provided by the sensor kinase, KinB, is required for this PhoB activation. We validated this relationship using both targeted and unbiased genetic approaches. eADAGE, which captures stable biological patterns, enables cross-experiment comparisons that can highlight measured but undiscovered relationships.

Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer. IMPORTANCE: Our understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system of P. aeruginosa Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that many P. aeruginosa strains maintain a CRISPR-Cas system.

Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis.

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.

Toxoplasma gondii seroprevalence in the United States 2009-2010 and comparison with the past two decades.

Toxoplasma gondii is a ubiquitous parasite that can cause neurologic and ocular disease. We tested sera from 7,072 people ≥ 6 years of age in the 2009-2010 National Health and Nutrition Examination Survey (NHANES) for immunoglobulin G antibodies and compared these results with two previous NHANES studies. The overall T. gondii antibody seroprevalence among persons ≥ 6 years of age in 2009-2010 was 13.2% (95% confidence limit [CL] 11.8%, 14.5%) and age-adjusted seroprevalence was 12.4% (95% CL 11.1%, 13.7%); age-adjusted seroprevalence among women 15-44 years of age was 9.1% (95% CL 7.2%, 11.1%). In U.S. born persons 12-49 years of age, the age-adjusted T. gondii seroprevalence decreased from 14.1% (95% CL 12.7%, 15.5%) in NHANES III (1988-1994) to 9.0% (95% CL 7.6%, 10.5%) in NHANES 1999-2004 to 6.7% (95% CL 5.3%, 8.2%) in NHANES 2009-2010 (P < 0.001 linear trend). Although T. gondii antibody presence is still relatively common, the prevalence in the United States has continued to decline.

A Phase 1 study of imatinib mesylate in combination with cytarabine and daunorubicin for c-kit positive relapsed acute myeloid leukemia.

The c-kit receptor is expressed in 95% of relapsed acute myeloid leukemias (AMLs) and mediates leukemic proliferation. We conducted a Phase 1 study of the c-kit inhibitor, imatinib mesylate (IM), in combination with cytarabine and daunorubicin (7+3) in c-kit+ relapsed AML. IM was dose escalated using a 3 by 3 design. Phosphorylated STAT5 was absent to minimally present in residual blasts on day 14 bone marrows. The maximum tolerated dose of IM was 300 mg. The dose-limiting toxicity was Grade 3-4 hepatic toxicity. The CR/CRp rate was 57%. Cytotoxic therapy that includes IM for relapsed AML is well-tolerated and effective.