Real-Time Identification of Pancreatic Cancer Cases Using Artificial Intelligence Developed on Danish Nationwide Registry Data.

PURPOSE: Pancreatic cancer is expected to be the second leading cause of cancer-related deaths worldwide within few years. Most patients are not diagnosed in time for curative-intent treatment. Accelerating the time of diagnosis is a key component of reducing pancreatic cancer mortality. We developed and tested a dynamic algorithm aiming at proactively identifying patients with a substantially elevated risk of having undiagnosed pancreatic cancer. METHODS: Machine learning methodology was applied to a live stream of nationwide Danish registry data. A hybrid case-control and prospective cohort design relying on incidence density sampling was used. Three models with minimal tuning were tested. All performance evaluation metrics were based on out-of-sample, out-of-time data in a monthly walk-forward strategy to avoid any temporal biases or inflation of performance metrics. Outcome was a diagnosis of pancreatic cancer. RESULTS: Subgroups identified had a 10.1% risk of being diagnosed with pancreatic cancer within 1 year, corresponding to a number needed to screen of 9.9. When considering competing, potentially computed tomography-detectable GI cancers, this number is reduced to 5.7. The time of diagnosis can be accelerated by up to 142 days. CONCLUSION: Currently available nationwide live data and computational resources are sufficient for real-time identification of individuals with at least 10.1% risk of having undiagnosed pancreatic cancer and 17.7% risk of any GI cancer in the Danish population. For prospective identification of high-risk patients, the area under the curve is not a useful indication of the positive predictive values achieved. Viable design solutions are demonstrated, which address the main shortfalls of the existing cancer prediction efforts in relation to temporal biases, leaks, and performance metric inflation. Efficacy evaluations with resection rates and mortality as end points are needed.

Isolation and characterization of multidrug resistant Gallibacterium anatis biovar haemolytica strains from Polish geese and hens.

Gallibacterium anatis biovar haemolytica is a bacterium that is frequently associated with infections of the reproductive tract and respiratory system in poultry. To assess the current prevalence and resistance profile of these bacteria in Poland, we collected and investigated 63 strains of Gallibacterium from diseased domestic poultry flocks including geese, laying hens, breeding hens and an ornamental hen. Detailed characterization of the isolates included the analysis of phenotypic antimicrobial resistance profiles and biofilm formation ability. Furthermore, the genetic background of 40 selected isolates regarding the presence of virulence and antimicrobial resistance genes and mobile genetic elements was determined. All investigated isolates were multidrug resistant, most prominently to ß-lactams, fluoroquinolones, sulfonamides and macrolides. A total of 48 different resistance profiles were detected. Of all isolates, 50.8% formed a strong biofilm, where strains isolated from geese appeared to be better at biofilm formation than strains isolated from laying and breeding hens. Single-nucleotide polymorphism genotyping revealed that G. anatis bv. haemolytica strains are restricted in host and geographical distribution, and the geese isolates showed greater phylogenetic similarity. Whole genome sequencing enabled identification of 25 different antimicrobial resistance determinants. The most common resistance genes were tetB, blaROB-1, and blaTEM-1 which may be located on mobile genetic elements. All isolates possessed the toxin gene gtxA, and the fimbrial gene flfA was identified in 95% of strains. Our results indicated that all G. anatis bv. haemolytica isolates showed multidrug resistant phenotypes. Strains isolated from geese were characterized by the highest percentage of isolates resistant to selected antimicrobials, probably reflecting host-related adaptations.

The effects of collaborative care versus consultation liaison for anxiety disorders and depression in Denmark: two randomised controlled trials.

BACKGROUND: Collaborative care (CC) and consultation liaison (CL) are two conceptual models aiming to improve mental healthcare in primary care. The effects of these models have not been compared in a Danish setting. AIMS: To examine the effects of CC versus CL for persons with anxiety and depression in Danish general practices (trial registration: NCT03113175 and NCT03113201). METHOD: Two randomised parallel superiority trials for anxiety disorders and depression were carried out in 2018-2019. In the CC-group, care managers collaborated with general practitioners (GPs) to provide evidence-based treatment according to structured treatment plans. They followed up and provided psychoeducation and/or cognitive-behavioural therapy. The GPs initiated pharmacological treatment if indicated, and a psychiatrist provided supervision. In the CL-group, the intervention consisted of the GP's usual treatment. However, the psychiatrist and care manager could be consulted. Primary outcomes were depression symptoms (Beck Depression Inventory-II, BDI-II) in the depression trial and anxiety symptoms (Beck Anxiety Inventory, BAI) in the anxiety trial at 6-month follow-up. RESULTS: In total, 302 participants with anxiety disorders and 389 participants with depression were included. A significant difference in BDI-II score was found in the depression trial, with larger symptom reductions in the CC-group (CC: 12.7, 95% CI 11.4-14.0; CL: 17.5, 95% CI 16.2-18.9; Cohen's d = -0.50, P ≤ 0.001). There was a significant difference in BAI in the anxiety trial (CC: 14.9, 95% CI 13.5-16.3; CL: 17.9, 95% CI 16.5-19.3; Cohen's d = -0.34, P ≤ 0.001), with larger symptom reductions in the CC-group. CONCLUSIONS: Collaborative care was an effective model to improve outcomes for persons with depression and anxiety disorders.

30-Month Follow-Up of Individual Placement and Support (IPS) and Cognitive Remediation for People with Severe Mental Illness: Results from a Randomized Clinical Trial.

Background: The individual placement and support (IPS) model for persons with severe mental illness has proven to be more effective than traditional vocational approaches in improving competitive work over 18 months. In this study, the longer-term effects of IPS over 30 months were investigated in a Danish setting. Method: In a randomized clinical trial, we compared the effects of IPS, IPS enhanced with cognitive remediation and work-related social skills training (IPSE), and service as usual (SAU). At three locations in Denmark, 720 patients with serious mental illnesses were randomly assigned to the three groups. Competitive employment, education, and hospital admissions were tracked for 30 months using Danish national registers. Results: The beneficial effects of IPS on competitive employment and education at the 18-month follow-up were sustained over the 30-month follow-up period. Participants receiving IPS or IPSE were more likely to obtain competitive employment or education than those who received service as usual (IPS 65%, IPSE 65%, SAU 53%, p = 0.006), and they worked on average more weeks competitively (IPS 25 weeks, IPSE 21 weeks, SAU 17 weeks; IPS vs. SAU p = 0.004 and IPSE vs. SAU p = 0.007). Moreover, participants in the two IPS groups had fewer outpatient visits during the 30-month follow-up. However, this was only statistically significant when comparing IPSE with SAU p = 0.017. Conclusion: In conclusion, IPS and IPS enhanced with cognitive remediation and work-related skills training demonstrated that the vocational effects of the interventions are retrained over 30 months in a Danish context.

Quinolone Resistance in Gallibacterium anatis Determined by Mutations in Quinolone Resistance-Determining Region.

Control of the important pathogen, Gallibacterium anatis, which causes salpingitis and peritonitis in poultry, relies on treatment using antimicrobial compounds. Among these, quinolones and fluoroquinolones have been used extensively, leading to a rise in the prevalence of resistant strains. The molecular mechanisms leading to quinolone resistance, however, have not previously been described for G. anatis, which is the aim of this study. The present study combines phenotypic antimicrobial resistance data with genomic sequence data from a collection of G. anatis strains isolated from avian hosts between 1979 and 2020. Minimum inhibitory concentrations were determined for nalidixic acid, as well as for enrofloxacin for each included strain. In silico analyses included genome-wide queries for genes known to convey resistance towards quinolones, identification of variable positions in the primary structure of quinolone protein targets and structural prediction models. No resistance genes known to confer resistance to quinolones were identified. Yet, a total of nine positions in the quinolone target protein subunits (GyrA, GyrB, ParC and ParE) displayed substantial variation and were further analyzed. By combining variation patterns with observed resistance patterns, positions 83 and 87 in GyrA, as well as position 88 in ParC, appeared to be linked to increased resistance towards both quinolones included. As no notable differences in tertiary structure were observed between subunits of resistant and sensitive strains, the mechanism behind the observed resistance is likely due to subtle shifts in amino acid side chain properties.

Predictors of return to work for people on sick leave with depression, anxiety and stress: secondary analysis from a randomized controlled trial.

PURPOSE: Knowledge about predictors of return to work (RTW) in people on sick leave with common mental disorders (CMDs) may inform the development of effective vocational rehabilitation interventions for this target group. In this study, we investigated predictors of RTW at 6 and 12 months in people on sick leave with depression, anxiety disorders or stress-related disorders. METHODS: We have performed a secondary analysis, utilizing data from two RCTs that evaluated the efficacy of an integrated health care and vocational rehabilitation intervention. Data were obtained from mental health assessments, questionnaires and registers. Using Cox regression analysis, the relationship between baseline variables and RTW was analysed at 6 and 12 months after randomization within the group of CMD as a whole and within the subgroups of depression, anxiety and stress-related disorders. RESULTS: Symptom burden and employment status at baseline predicted RTW in the CMD group (n = 1245) and in the three diagnostic subgroups at both time points. RTW self-efficacy predicted RTW in the depression group but not in the anxiety or stress subgroups. CONCLUSION: Many predictors of RTW were similar over time and, to some extent, across the CMD subgroups. Findings highlight the need not only to take health-related and psychological factors into account when developing vocational rehabilitation interventions but also to consider workplace strategies and options for support.

Risk Factors, Genetic Diversity, and Antimicrobial Resistance of Staphylococcus spp. Isolates in Dogs Admitted to an Intensive Care Unit of a Veterinary Hospital.

Intensive Care Units (ICU) usually provide an excellent environment for the selection of pathogens associated with hospital-acquired infections (HAI), leading to increased mortality and hospitalization costs. Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of HAI in dogs worldwide, but the risk factors and dynamics of colonization by MRSP are largely unknown. This study aimed to evaluate the risk factors associated with the acquisition of MRSP in dogs admitted to an ICU, and to report the antimicrobial resistance profiles and genetic relatedness of MRSP isolates. Sterile swabs from the nostril, axilla, and rectum were collected daily during the hospitalization of 54 dogs. Samples were subjected to Mannitol Salt Agar, and colonies were identified by MALDI-ToF, polymerase chain reaction (PCR), and sequencing of the rpoB gene. Antimicrobial susceptibility testing and PCR detection of mecA were performed. Staphylococcus spp. was isolated from 94% of the dogs, and the most frequently isolated species was S. pseudintermedius (88.2%). Carriage of multidrug resistant (MDR) staphylococci was observed in 64.4% of the dogs, and approximately 39% had methicillin-resistant Staphylococcus sp. (MRS), of which 21.6% had MRSP and 1.9% had methicillin-resistant S. aureus (MRSA). The acquisition of MRSP during ICU hospitalization was associated with sex (female), age (>7 years), and dogs that had previously been treated with antimicrobials. Animals colonized by MRSP resistant to ≥9 antimicrobial classes had longer hospital stays than those colonized by other MRS strains. Among the 13 MRSP isolates that were subjected to whole-genome sequencing, ten were classified as ST71. A single nucleotide polymorphism (SNP) analysis revealed three clones, including one that was detected in infected dogs outside the ICU. This study indicates novel risk factors associated with colonization by MRSP. The detection of the same MRSP clone causing HAI outside the ICU reinforces the need for improved infection prevention and control practices at veterinary hospitals in general and at the ICU in particular.

Dramatic increase in slaughter condemnations due to Escherichia coli ST23 and ST101 within the Danish broiler production.

Escherichia coli constitutes a major challenge to poultry even when the prevalence of colibacillosis is low. Additionally, specific E. coli strains can severely enhance the detrimental effects on productivity, animal welfare and antimicrobial use. In 2019-2020, a dramatic increase in colibacillosis occurred among Danish broilers causing late-onset mortality and high slaughter condemnations. In the present study, the pathology and causative E. coli-types were characterised. Furthermore, the outbreak-related strains were compared to isolates from concurrent "background" colibacillosis. During the study, 1039 birds were subjected to a comprehensive post-mortem examination, and a total of 349 E. coli isolates were sequenced and characterised by multi-locus sequence typing, virulence and resistance gene presence, plasmid replicon content and phylogenetic analysis. Productivity data from outbreak flocks revealed a mortality of 6.34% ± 3.74 and a condemnation of 5.04% ± 3.67. Contrary, the numbers were 3.18% ± 1.57% and 1.02% ± 0.4 among non-outbreak flocks, respectively. Major lesions were cellulitis (46.82%), airsacculitis (67.63%), pericarditis (55.49%), perihepatitis (41.04%) and femoral head necrosis with physeal/metaphyseal involvement (44.51%). Among non-outbreak broilers, the prevalence was 4.46%, 7.64%, 7.01%, 3.82% and 8.28%, respectively. ST23 and ST101 dominated heavily in outbreak flocks, whereas non-outbreak related isolates consisted of various other STs. A low level of resistance markers was evident, except in few multidrug-resistant isolates. Within ST23 and ST101, 13 and 12 virulence genes were significantly over-represented compared to non-outbreak isolates. In conclusion, clonal lineages were documented as the cause of a devastating outbreak of colibacillosis with great prospects for future interventions.

Integrated mental healthcare and vocational rehabilitation for people on sick leave with stress-related disorders: 24-month follow-up of the randomized IBBIS trial.

OBJECTIVES: Integrating vocational rehabilitation and mental healthcare has shown effects on vocational outcomes during sick leave with common mental disorders. In a previous paper, we showed that a Danish integrated healthcare and vocational rehabilitation intervention (INT) had a surprisingly negative impact on vocational outcomes compared to service as usual (SAU) at 6- and 12-month follow-up. That was also the case with a mental healthcare intervention (MHC) tested in the same study. This article reports the 24-month follow-up results of that same study. METHOD: A randomized, parallel-group, three-arm, multi-centre superiority trial was conducted to test the effectiveness of INT and MHC compared to SAU. RESULTS: In total, 631 persons were randomized. Contrary to our hypothesis, SAU showed faster return to work than both INT [hazard rate (HR) 1.39, P=0.0027] and MHC (HR 1.30, P=0.013) at 24-month follow-up. Overall, no differences were observed regarding mental health and functional level. Compared to SAU, we observed some health benefits of MHC, but not INT, at 6-month follow-up but not thereafter, and lower rates of employment at all follow-ups. Since implementation problems might explain the results of INT, we cannot conclude that INT is no better that SAU. The MHC intervention was implemented with good fidelity and did not improve return to work. CONCLUSION: This trial does not support the hypothesis that INT lead to faster return to work. However, implementation failure may explain the negative results.

Integrated Mental Healthcare and Vocational Rehabilitation for People on Sick Leave with Anxiety or Depression: 24-Month Follow-up of the Randomized IBBIS Trial.

Integration of vocational rehabilitation and mental healthcare has shown some effect on work participation at 1-year follow-up after sick leave with depression and anxiety. We aimed to study the effect on work and health outcomes at 2-year follow-up, why we performed a randomized trial was conducted to study the effectiveness of integrated intervention (INT) compared to service as usual (SAU) and best practice mental healthcare (MHC). We included 631 participants, and at 24-month follow-up, we detected no differences in effect between INT and SAU. Compared to MHC, INT showed faster return-to-work (RTW) rates (p = 0.044) and a higher number of weeks in work (p = 0.024). No symptom differences were observed between the groups at 24 months. In conclusion, compared to SAU, INT was associated with a slightly higher work rate reaching borderline statistical significance at 12-month follow-up and lower stress levels at 6-month follow-up. The disappearance of relative effect between 12 and 24 months may be explained by the fact that the intervention lasted less than 12 months or by delayed spontaneous remission in the SAU group after 12 months. Despite the lack of effect at long-term follow-up, INT still performed slightly better than SAU overall. Moderate implementation difficulties, may partly explain the absence of the hypothesized effect. Integrated intervention, as implemented in this trial, showed some positive effects on mid-term vocational status and short-term stress symptom levels. However, these effects were not sustained beyond the duration of the intervention.

Predictors of Return to Work for People with Anxiety or Depression Participating in a Randomized Trial Investigating the Effect of a Supported Employment Intervention.

Purpose Common mental disorders have a severe impact on society and individuals; rates of unemployment and disability pensions are high. Knowing which factors facilitate or hinder people's return to work is important when designing effective vocational rehabilitation interventions. Methods We conducted secondary analyses on data from 289 participants with depression or anxiety included in the Individual Placement and Support modified for people with mood and anxiety disorders (IPS-MA) trial. Associations of baseline characteristics and employment or education after 24 months were tested in univariate logistic regression analyses, variables with a p-value below 0.1 were included in multivariate analyses. Results In the univariate analyses, self-reported level of functioning (p = 0.032), higher age (p = 0.070), and higher level of readiness to change (p = 0.001) were associated with the outcome and included in the multivariate analysis. Only age (p = 0.030) and readiness to change (p = 0.003) remained significantly associated with return to work or education after 24 months in the multivariate analysis. Conclusion Higher age and lower readiness to change were associated with a lower chance of having returned to work or education. Factors modifying the effect of higher age should be identified, just as vocational rehabilitation should focus on improving factors related to people's readiness to change.

Addressing a Rule of Thumb: Modeling the Effects of Meteorological Conditions on Prescription of Antimicrobials in Aquaculture.

Meteorological phenomena such as warm summer temperatures and increased numbers of sunlight hours have repeatedly been hypothesized to be associated with infectious diseases and an increased prescription of antimicrobial compounds in Danish aquaculture. To address this hypothesis, we prepared statistical models incorporating aquaculture production data from Danish Aquaculture, detailed records on prescription of antimicrobials from the Danish VETSTAT program, and meteorological data from 2001 to 2019 from the Danish Meteorological Institute. Separate series of models were made and refined for land-based and marine production, respectively. For both production forms, the models identify summer sunlight hours as having a significant influence on antimicrobial use. In addition to summer sunlight, spring sunlight and water temperature were integral, although not statistically significant, parameters when modeling antimicrobial use in marine production. Although the extensive availability and accuracy of relevant data are associated with Danish production, we believe the results allow for more general conclusions on the influence of meteorological parameters on outbreaks of bacterial pathogens in international aquaculture. Such insights could have a substantial impact on prophylactic strategies, fish husbandry, and our understanding of how increasing temperatures may affect future antimicrobial usage in the global aquaculture industry. IMPORTANCE Global aquaculture production has been rapidly increasing for decades and is set to play a pivotal role in feeding a growing human population. Along with the growth in aquaculture production, the annual global use of antimicrobials is estimated to increase by one-third between 2017 and 2030. Using detailed antimicrobial prescription records as a proxy for outbreaks, we were able to evaluate the effects of a variety of meteorological parameters through statistical modeling. Our results lend scientific support to informal observations from the industry, but more importantly, this study provides novel, essential information on the importance of abiotic factors that can, in turn, lead to improved prophylactic efforts and thus help to reduce antimicrobial use in global aquaculture.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Longitudinal study on background lesions in broiler breeder flocks and their progeny, and genomic characterisation of Escherichia coli.

In broiler breeders, background mortality is rarely addressed, however, it represents the death of a vast number of birds, a constant productivity loss, welfare concerns and it might affect chick quality. The study aimed to unveil lesions leading to mortality in a study population perceived as healthy, combined with whole-genome sequencing (WGS) of Escherichia coli, a well-known contributor to disease problems in poultry. Broiler breeders (n = 340) originating from three distinct, putative healthy flocks and their progeny (n = 154) were subjected to a comprehensive post-mortem examination, bacteriological sampling, and sequencing of 77 E. coli isolates. Productivity data confirmed an exemplary health status of the enrolled flocks, and post-mortem examination further verified the absence of general disease problems. Among the submitted broiler breeders, exudative peritonitis (31.2%) was the most frequent lesion linked to infectious disease, whereas airsacculitis, pericarditis, perihepatitis, and salpingitis occurred in 18.5%, 3.5%, 3.8% and 17%, respectively. Yolksacculitis occurred in 15.6% of the broilers, whilst pericarditis, perihepatitis and peritonitis were diagnosed in 9.7%, 7.1% and 9.1%, respectively. WGS revealed a diverse population where ST95 dominated the population retrieved from broiler breeders, whereas ST10 was highly prevalent among broilers. Both lineages could be isolated from extraintestinal sites of birds without lesions indicative of infection. In general, the genetic diversity within flocks was comparable to the diversity between farms, and the overall occurrence of resistance markers was low. In conclusion, a comprehensive insight into lesions associated with background mortality is presented, together with a vast diversity of E. coli isolated from extraintestinal sites during a non-outbreak situation.

Trends in pancreatic cancer incidence, characteristics, and outcomes in Denmark 1980-2019: A nationwide cohort study.

OBJECTIVE: To describe time-trends in incidence, characteristics, treatments, and survival in pancreatic cancer patients in Denmark during 1980-2019. DESIGN: A nationwide population-based cohort study of all Danish patients diagnosed with exocrine pancreatic cancer during the study period. Data was obtained from individual-level cross linkage between Danish healthcare registries. We present descriptive characteristics and survival estimates, which was obtained using the Kaplan-Meier estimator and Cox proportional hazards regression models. RESULTS: During the study period, 32,107 patients were diagnosed with pancreatic cancer. In the most recent period, the age-standardized incidence rate was 17.7 per 100,000 person-years. Throughout the study period, between 18.4% and 27.5% of patients had no tumor staging performed, and approximately half of the patient were only offered best supportive care. The proportion of patients treated with surgery doubled during the study period, and the use of adjuvant and neoadjuvant oncological therapy increased substantially. Median survival after surgical resection also increased to 25.8 months in the most recent time period. CONCLUSION: Pancreatic cancer incidence is increasing in Denmark, and this increase is projected to continue. The proportion of patients offered curative-intent treatment increased, which translates into an increase in overall survival. All numbers are comparable to best international standards.

Clarification of large-volume bacterial cultures using a centrifuge-free protocol.

AIMS: To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures. METHODS AND RESULTS: Four experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS-PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge-free filtrates contained an increased amount of OMVs compared to centrifuged filtrates. CONCLUSIONS: In comparison to conventional centrifuge-based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge-free approach. SIGNIFICANCE AND IMPACT OF THE STUDY: Sterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a novel approach in improving the filtrate products without the need of centrifugation.

Fecal Shedding of Multidrug Resistant Escherichia coli Isolates in Dogs Fed with Raw Meat-Based Diets in Brazil.

The practice of feeding dogs raw meat-based diets (RMBDs) is growing in several countries, and the risks associated with the ingestion of pathogenic and antimicrobial-resistant Escherichia coli in dogs fed these diets are largely unknown. We characterized E. coli strains isolated from dogs fed either an RMBD or a conventional dry feed, according to the phylogroup, virulence genes, and antimicrobial susceptibility profiles of the bacteria. Two hundred and sixteen E. coli strains were isolated. Dogs fed RMBDs shed E. coli strains from the phylogroup E more frequently and were positive for the E. coli heat-stable enterotoxin 1-encoding gene. Isolates from RMBD-fed dogs were also frequently positive for multidrug-resistant E. coli isolates including extended-spectrum beta-lactamase (ESBL) producers. Whole-genome sequencing of seven ESBL-producing E. coli strains revealed that they predominantly harbored blaCTX-M-55, and two strains were also positive for the colistin-resistant gene mcr-1. These results suggest that feeding an RMBD can affect the dog's microbiota, change the frequency of certain phylogroups, and increase the shedding of diarrheagenic E. coli. Also, feeding an RMBD seemed to be linked with the fecal shedding of multidrug-resistant E. coli, including the spread of strains harboring mobilizable colistin resistance and ESBL genes. This finding is of concern for both animal and human health.

Integrating vocational rehabilitation and mental healthcare to improve the return-to-work process for people on sick leave with stress-related disorders: results from a randomized trial.

OBJECTIVE: Stress-related disorders are common, associated with substantial individual suffering, and place a large economic burden on society. While treatment appears to be able to reduce symptoms, evidence of interventions to improve vocational outcomes is flimsy. Lack of integration of vocational rehabilitation and healthcare services has been suspected to be a major potential barrier in return-to-work (RTW) processes; therefore, we aimed to test the effectiveness of such integration. METHODS: We randomized participants who were on sick leave for ≥ 4 weeks with a stress-related disorder. They were allocated to (i) service as usual (SAU), (ii) improved mental healthcare (MHC), or (iii) integrated interventions (INT). The primary outcome was RTW rates measured at 12 months. Secondary outcome were RTW rates measured at 6 months, proportion in work at 12 months, and levels of stress, anxiety, depression, and functioning at 6 months. RESULTS: We included 666 participants. On the primary outcome and almost all other vocational outcomes, SAU was superior to both INT and MHC. MHC and INT did not differ on any vocational outcome. On several symptom scales, MHC showed lower values than SAU, whilst INT did not differ from the two other groups. CONCLUSION: Both the INT and the MHC intervention lowered RTW rates compared with SAU, and thereby yielded a worse outcome. However, the MHC group showed a tendency towards having lower symptom levels compared with those in the SAU group; accordingly, the SAU group is not unequivocally superior. MHC and INT showed no general differences.

Reduced fibrin clot lysis in Klinefelter syndrome associated with hypogonadism.

Objective: Klinefelter syndrome (KS) is associated with increased risk of thrombosis. Hypogonadism and accumulating body fat in KS have a potential impact on fibrinolysis. In this study, we assessed the fibrinolytic system and the association with testosterone levels in KS. Design: This study is a cross-sectional comparison of men with KS and age-matched male controls. Methods: Fibrin clot lysis was evaluated by turbidity measurements and by measuring levels of individual fibrinolytic proteins in plasma samples. Fibrin clot structure was evaluated by scanning electron microscopy. Total testosterone was measured by liquid chromatography-tandem mass spectrometry. Body fat was evaluated by dual-energy X-ray absorptiometry. Results: In this study, 45 men with KS and 45 age- and education-matched controls were included. Men with KS had a 24% reduction in fibrin clot lysis compared with controls (46.2 ± 17.1 vs 60.6 ± 18.8 %/h, P = 0.0003) and higher levels of fibrinogen, factor XIII (P ≤ 0.01), and plasminogen activator inhibitor type 1 (P = 0.04). Men with KS had lower total testosterone (P = 0.008) and higher body fat (P = 0.001). In KS, reduced fibrin clot lysability was associated with higher fibrinogen and body fat related to decreasing total testosterone and hypogonadism among men with KS. Fibrin clot structure was not different compared to KS and controls. Conclusions: Fibrin clot lysis in KS was markedly reduced, potentially contributing to a prothrombotic state and increasing thrombotic risk. Hypogonadism in KS was associated with increased fibrinogen and total body fat, predicting reduced fibrin clot lysis.

Assessment of automated assays for serum amyloid A, haptoglobin (PIT54) and basic biochemistry in broiler breeders experimentally infected with Escherichia coli.

Biomarkers of inflammation are valuable tools for health status evaluation in numerous species. However, in poultry, methods for measuring acute phase proteins (APP) are sparse and rely on manual laboratory labour reserving these parameters mainly for research studies with APP as a focus point. To extend the use of APP beyond tightly focused research studies, blood from experimentally infected and control hens was analysed using equipment available in many veterinary clinics in order to identify easily accessible biomarkers of infection. Blood samples from broiler breeders (n = 30) inoculated intratracheally with either Escherichia coli or sterile vehicle were randomly selected at 2, 4 and 7 days post-infection (dpi) and subjected to biochemical analysis. Samples for bacteriological testing were collected, and all animals were subjected to a full necropsy for disease confirmation. Significantly higher levels of serum amyloid A were evident in the infected birds at 2 and 4 dpi (p < 0.01) compared to the controls. Likewise, haptoglobin (PIT54) levels were significantly elevated at 4 dpi (p < 0.01) in the infected animal, whilst at 2 dpi magnesium and calcium were significantly lower in the infected group (p < 0.05). Gross pathology and bacteriology confirmed the presence of infection in the E. coli inoculated birds. In conclusion, equipment routinely used in other species for rapid analysis of blood samples, successfully differentiated between sick and healthy birds, hereby, showing great potential as an easily added parameter of evaluation in research studies, and as a valuable decision-making tool for poultry veterinarians.