Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

<p>Abstract</p> <p>Background</p> <p>Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium <it>Dichelobacter nodosus</it>. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but <it>D. nodosu</it>s should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of <it>D. nodosus </it>and to compare its performance with culturing and conventional PCR.</p> <p>Methods</p> <p>A <it>D. nodosus-</it>specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.</p> <p>Results</p> <p>The developed assay had a detection limit of 3.9 fg of <it>D. nodosus </it>genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the <it>D. nodosus </it>genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.</p> <p>Conclusions</p> <p>The developed real-time PCR assay has good specificity and sensitivity for detection of <it>D. nodosus</it>, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.</p

Sites of persistence of Fusobacterium necrophorum and Dichelobacter nodosus: a paradigm shift in understanding the epidemiology of footrot in sheep

Sites of persistence of bacterial pathogens contribute to disease dynamics of bacterial diseases. Footrot is a globally important bacterial disease that reduces health and productivity of sheep. It is caused by Dichelobacter nodosus, a pathogen apparently highly specialised for feet, while Fusobacterium necrophorum, a secondary pathogen in footrot is reportedly ubiquitous on pasture. Two prospective longitudinal studies were conducted to investigate the persistence of D. nodosus and F. necrophorum in sheep feet, mouths and faeces, and in soil. Molecular tools were used to detect species, strains and communities. In contrast to the existing paradigm, F. necrophorum persisted on footrot diseased feet, and in mouths and faeces; different strains were detected in feet and mouths. D. nodosus persisted in soil and on diseased, but not healthy, feet; similar strains were detected on both healthy and diseased feet of diseased sheep. We conclude that D. nodosus and F. necrophorum depend on sheep for persistence but use different strategies to persist and spread between sheep within and between flocks. Elimination of F. necrophorum would be challenging due to faecal shedding. In contrast D. nodosus could be eliminated if all footrot-affected sheep were removed and fade out of D. nodosus occurred in the environment before re-infection of a foot

A distinct bacterial dysbiosis associated skin inflammation in ovine footrot

Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1β and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression

Be careful with triage in emergency departments: interobserver agreement on 1,578 patients in France

<p>Abstract</p> <p>Background</p> <p>For several decades, emergency departments (EDs) utilization has increased, inducing ED overcrowding in many countries. This phenomenon is related partly to an excessive number of nonurgent patients. To resolve ED overcrowding and to decrease nonurgent visits, the most common solution has been to triage the ED patients to identify potentially nonurgent patients, i.e. which could have been dealt with by general practitioner. The objective of this study was to measure agreement among ED health professionals on the urgency of an ED visit, and to determine if the level of agreement is consistent among different sub-groups based on following explicit criteria: age, medical status, type of referral to the ED, investigations performed in the ED, and the discharge from the ED.</p> <p>Methods</p> <p>We conducted a multicentric cross-sectional study to compare agreement between nurses and physicians on categorization of ED visits into urgent or nonurgent. Subgroups stratified by criteria characterizing the ED visit were analyzed in relation to the outcome of the visit.</p> <p>Results</p> <p>Of 1,928 ED patients, 350 were excluded because data were lacking. The overall nurse-physician agreement on categorization was moderate (kappa = 0.43). The levels of agreement within all subgroups were variable and low. The highest agreement concerned three subgroups of complaints: cranial injury (kappa = 0.61), gynaecological (kappa = 0.66) and toxicology complaints (kappa = 1.00). The lowest agreement concerned two subgroups: urinary-nephrology (kappa = 0.09) and hospitalization (kappa = 0.20). When categorization of ED visits into urgent or nonurgent cases was compared to hospitalization, ED physicians had higher sensitivity and specificity than nurses (respectively 94.9% versus 89.5%, and 43.1% versus 30.9%).</p> <p>Conclusions</p> <p>The lack of physician-nurse agreement and the inability to predict hospitalization have important implications for patient safety. When urgency screening is used to determine treatment priority, disagreement might not matter because all patients in the ED are seen and treated. But using assessments as the basis for refusal of care to potential nonurgent patients raises legal, ethical, and safety issues. Managed care organizations should be cautious when applying such criteria to restrict access to EDs.</p

Crop modelling: towards locally relevant and climate-informed adaptation

A gap between the potential and practical realisation of adaptation exists: adaptation strategies need to be both climate-informed and locally relevant to be viable. Place-based approaches study local and contemporary dynamics of the agricultural system, whereas climate impact modelling simulates climate-crop interactions across temporal and spatial scales. Crop-climate modelling and place-based research on adaptation were strategically reviewed and analysed to identify areas of commonality, differences, and potential learning opportunities to enhance the relevance of both disciplines through interdisciplinary approaches. Crop-modelling studies have projected a 7–15% mean yield change with adaptation compared to a non-adaptation baseline (Nature Climate Change 4:1–5, 2014). Of the 17 types of adaptation strategy identified in this study as place-based adaptations occurring within Central America, only five were represented in crop-climate modelling literature, and these were as follows: fertiliser, irrigation, change in planting date, change in cultivar and area cultivated. The breath and agency of real-life adaptation compared to its representation in modelling studies is a source of error in climate impact simulations. Conversely, adaptation research that omits assessment of future climate variability and impact does not enable to provide sustainable adaptation strategies to local communities so risk maladaptation. Integrated and participatory methods can identify and reduce these sources of uncertainty, for example, stakeholder’s engagement can identify locally relevant adaptation pathways. We propose a research agenda that uses methodological approaches from both the modelling and place-based approaches to work towards climate-informed locally relevant adaptation

The T7-Related Pseudomonas putida Phage ϕ15 Displays Virion-Associated Biofilm Degradation Properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy

COVID-19 trajectories among 57 million adults in England: a cohort study using electronic health records

BACKGROUND: Updatable estimates of COVID-19 onset, progression, and trajectories underpin pandemic mitigation efforts. To identify and characterise disease trajectories, we aimed to define and validate ten COVID-19 phenotypes from nationwide linked electronic health records (EHR) using an extensible framework. METHODS: In this cohort study, we used eight linked National Health Service (NHS) datasets for people in England alive on Jan 23, 2020. Data on COVID-19 testing, vaccination, primary and secondary care records, and death registrations were collected until Nov 30, 2021. We defined ten COVID-19 phenotypes reflecting clinically relevant stages of disease severity and encompassing five categories: positive SARS-CoV-2 test, primary care diagnosis, hospital admission, ventilation modality (four phenotypes), and death (three phenotypes). We constructed patient trajectories illustrating transition frequency and duration between phenotypes. Analyses were stratified by pandemic waves and vaccination status. FINDINGS: Among 57 032 174 individuals included in the cohort, 13 990 423 COVID-19 events were identified in 7 244 925 individuals, equating to an infection rate of 12·7% during the study period. Of 7 244 925 individuals, 460 737 (6·4%) were admitted to hospital and 158 020 (2·2%) died. Of 460 737 individuals who were admitted to hospital, 48 847 (10·6%) were admitted to the intensive care unit (ICU), 69 090 (15·0%) received non-invasive ventilation, and 25 928 (5·6%) received invasive ventilation. Among 384 135 patients who were admitted to hospital but did not require ventilation, mortality was higher in wave 1 (23 485 [30·4%] of 77 202 patients) than wave 2 (44 220 [23·1%] of 191 528 patients), but remained unchanged for patients admitted to the ICU. Mortality was highest among patients who received ventilatory support outside of the ICU in wave 1 (2569 [50·7%] of 5063 patients). 15 486 (9·8%) of 158 020 COVID-19-related deaths occurred within 28 days of the first COVID-19 event without a COVID-19 diagnoses on the death certificate. 10 884 (6·9%) of 158 020 deaths were identified exclusively from mortality data with no previous COVID-19 phenotype recorded. We observed longer patient trajectories in wave 2 than wave 1. INTERPRETATION: Our analyses illustrate the wide spectrum of disease trajectories as shown by differences in incidence, survival, and clinical pathways. We have provided a modular analytical framework that can be used to monitor the impact of the pandemic and generate evidence of clinical and policy relevance using multiple EHR sources. FUNDING: British Heart Foundation Data Science Centre, led by Health Data Research UK