The COVID-19 ambulance response assessment (CARA) study: a national survey of ambulance service healthcare professionals' preparedness and response to the COVID-19 pandemic.

Background: The COVID-19 pandemic placed significant demand on the NHS, including ambulance services, but it is unclear how this affected ambulance service staff and paramedics in other clinical settings (e.g. urgent and primary care, armed services, prisons). This study aimed to measure the self-perceived preparedness and impact of the first wave of the pandemic on paramedics' psychological stress and perceived ability to deliver care. Methods: Ambulance clinicians and paramedics working in other healthcare settings were invited to participate in a three-phase sequential online survey during the acceleration (April 2020), peak (May 2020) and deceleration (September/October 2020) phases of the first wave of COVID-19 in the United Kingdom. Recruitment used social media, Trust internal bulletins and the College of Paramedics' communication channels, employing a convenience sampling strategy. Data were collected using purposively developed open- and closed-ended questions and the validated general health questionnaire-12 (GHQ-12). Data were analysed using multi-level linear and logistic regression models. Results: Phase 1 recruited 3717 participants, reducing to 2709 (73%) by phase 2 and 2159 (58%) by phase 3. Participants were mostly male (58%, n = 2148) and registered paramedics (n = 1992, 54%). Mean (standard deviation) GHQ-12 scores were 16.5 (5.2) during phase 1, reducing to 15.2 (6.7) by phase 3. A total of 84% of participants (n = 3112) had a GHQ-12 score ≥ 12 during the first phase, indicating psychological distress. Participants that had higher GHQ-12 scores were feeling unprepared for the pandemic, and reported a lack of confidence in using personal protective equipment and managing cardiac arrests in confirmed or suspected COVID-19 patients. Conclusions: Most participants reported psychological distress, the reasons for which are multi-factorial. Ambulance managers need to be aware of the risks to staff mental health and take action to mitigate these, to support staff in the delivery of unscheduled, emergency and urgent care under these additional pressures.

Is there an association between 30-day mortality and adrenaline infusion rates in post-ROSC patients? A retrospective observational analysis.

Introduction: Revised guidelines for the management of cardiac arrest have placed greater emphasis on early defibrillation and closed chest compressions; subsequently there has been a significant rise in the number of patients gaining a return of spontaneous circulation (ROSC). As a consequence, emergency medical services have realised the importance of therapies delivered during this phase of care. In some Trusts this includes the use of inotropic agents to augment the cardiovascular system and maintain adequate cerebral and coronary perfusion pressures to mitigate the effects of post-cardiac arrest syndrome. Currently, limited evidence exists with regards to the efficacy of such treatments in the pre-hospital phase. Methods: Retrospective observational analysis of out-of-hospital cardiac arrest patients who received an adrenaline infusion by critical care paramedics. Infusion rates, time of call (ToC) to ROSC and 30-day mortality were compared. Results: Over a 2-year period, 202 patients were recorded as having an adrenaline infusion commenced. Of these, 25 were excluded as they did not meet criteria or had incomplete data and 22 were excluded as the infusion was stopped at scene; 155 patients were admitted to hospital. There were no survivors in the non-shockable group and three survivors in the shockable group at 30 days. A rare events analysis found no relationship between infusion rate, ToC to ROSC and 30-day mortality (Wald chi2, 1.37). Conclusion: Commencement of adrenaline infusions in post-ROSC was associated with significant 30-day mortality, especially in non-shockable rhythms. Further research is needed to elucidate whether this intervention has any benefit in the post-ROSC patient.

Initiating an undiagnosed diseases program in the Western Australian public health system.

BACKGROUND: New approaches are required to address the needs of complex undiagnosed diseases patients. These approaches include clinical genomic diagnostic pipelines, utilizing intra- and multi-disciplinary platforms, as well as specialty-specific genomic clinics. Both are advancing diagnostic rates. However, complementary cross-disciplinary approaches are also critical to address those patients with multisystem disorders who traverse the bounds of multiple specialties and remain undiagnosed despite existing intra-specialty and genomic-focused approaches. The diagnostic possibilities of undiagnosed diseases include genetic and non-genetic conditions. The focus on genetic diseases addresses some of these disorders, however a cross-disciplinary approach is needed that also simultaneously addresses other disorder types. Herein, we describe the initiation and summary outcomes of a public health system approach for complex undiagnosed patients - the Undiagnosed Diseases Program-Western Australia (UDP-WA). RESULTS: Briefly the UDP-WA is: i) one of a complementary suite of approaches that is being delivered within health service, and with community engagement, to address the needs of those with severe undiagnosed diseases; ii) delivered within a public health system to support equitable access to health care, including for those from remote and regional areas; iii) providing diagnoses and improved patient care; iv) delivering a platform for in-service and real time genomic and phenomic education for clinicians that traverses a diverse range of specialties; v) retaining and recapturing clinical expertise; vi) supporting the education of junior and more senior medical staff; vii) designed to integrate with clinical translational research; and viii) is supporting greater connectedness for patients, families and medical staff. CONCLUSION: The UDP-WA has been initiated in the public health system to complement existing clinical genomic approaches; it has been targeted to those with a specific diagnostic need, and initiated by redirecting existing clinical and financial resources. The UDP-WA supports the provision of equitable and sustainable diagnostics and simultaneously supports capacity building in clinical care and translational research, for those with undiagnosed, typically rare, conditions.

The efficacy of disinfectants in the decontamination of dental unit water lines: an in vitro laboratory study.

OBJECTIVES/AIMS: This in vitro laboratory study compared the efficacy of water, sodium percarbonate (SPC) and chlorine dioxide (ClO2) solutions in the disinfection of dental unit water lines (DUWLs). MATERIALS AND METHODS: New DUWL tubes were cut, split open, and mono-culture and mixed-culture biofilms of Staphylococcus aureus, Enterococcus faecalis and Streptococcus mutans were grown. Harvested biofilms from the sectioned DUWL tubes were exposed to sterile distilled water, SPC or 5 and 10 p.p.m. ClO2 in both a stationary phase and through a constant flow. Bacterial counts were compared using the Kruskal-Wallis nonparametric rank test. RESULTS: In the mono-culture biofilms, SPC, 5 and 10 p.p.m. ClO2 significantly reduced all the test organisms (P<0.01). However, no significant difference was found between SPC and ClO2. In the mixed-culture biofilms exposed to disinfectant without flow, ClO2 significantly reduced the biofilm (P=0.02) compared with water and SPC. Similarly, in the constant flow study, ClO2 proved to be superior to water. CONCLUSION: At low concentrations, ClO2 with and without flow significantly reduced the mixed-culture biofilm grown in vitro on the sections of the DUWL tubes. Therefore, it has the potential to be used in the patient treatment water, as it is potable at these concentrations, and to decontaminate and limit the biofilm formation in the water lines.

A comparison of the accuracy of polyether, polyvinyl siloxane, and plaster impressions for long-span implant-supported prostheses.

PURPOSE: The purpose of this study was to compare the capacity of different impression materials to accurately reproduce the positions of five implant analogs on a master model by comparing the resulting cast with the stainless steel master model. The study was motivated by the knowledge that distortions can occur during impression making and the pouring of casts and that this distortion may produce inaccuracies of subsequent restorations, especially long-span castings for implant superstructures. MATERIALS AND METHODS: The master model was a stainless steel model with five implant analogs. The impression materials used were impression plaster (Plastogum, Harry J Bosworth), a polyether (Impregum Penta, 3M ESPE), and two polyvinyl siloxane (PVS) materials (Aquasil Monophase and Aquasil putty with light-body wash, Dentsply). Five impressions were made with each impression material and cast in die stone under strictly controlled laboratory conditions. The positions of the implants on the master model, the impression copings, and the implant analogs in the subsequent casts were measured using a coordinate measuring machine that measures within 4 µm of accuracy. RESULTS: Statistical analyses indicated that distortion occurred in all of the impression materials, but inconsistently. The PVS monophase material reproduced the master model most accurately. Although there was no significant distortion between the impressions and the master model or between the impressions and their casts, there were distortions between the master model and the master casts, which highlighted the cumulative effects of the distortions. The polyether material proved to be the most reliable in terms of predictability. The impression plaster displayed cumulative distortion, and the PVS putty with light body showed the least reliability. CONCLUSIONS: Some of the distortions observed are of clinical significance and likely to contribute to a lack of passive fit of any superstructure. The inaccuracy of these analog materials and procedures suggested that greater predictability may lie in digital technology.

Characterization of the haem-uptake system of the equine pathogen Streptococcus equi subsp. equi.

Streptococcus equi possesses a haem-uptake system homologous to that of Streptococcus pyogenes and Streptococcus zooepidemicus. The system consists of two ligand-binding proteins (Shr and Shp) and proteins (HtsA-C) with homology to an ABC transporter. The haem-uptake system of S. equi differs from that of S. pyogenes and S. zooepidemicus in that Shr is truncated by two-thirds. This study focused on the SeShr, SeShp and SeHtsA proteins of S. equi. Analysis of shr, shp and shphtsA knockout mutants showed that all three proteins were expressed in vitro and that expression was upregulated under conditions of iron limitation. SeShr possesses no membrane-/cell wall-spanning sequences and was shown to be secreted. Both SeShp and SeHtsA were confirmed to be envelope-associated. Recombinant SeShp and SeHtsA proteins have been previously shown to bind haem and SeHtsA could capture haem from SeShp. This report extends these studies and shows that both SeShp and SeHtsA can sequester haem from haemoglobin but not from haemoglobin-haptoglobin complexes. Like full-length Shr, SeShr possesses haemoglobin and haemoglobin-haptoglobin binding ability but unlike full-length Shr, it lacks haem- or fibronectin-binding capabilities. Analysis of SeShr truncates showed that residues within and upstream of the near transporter (NEAT) domain are required for this ligand binding. Structural predictions suggest that truncation of NEAT1 in SeShr accounts for its impaired ability to bind haem. Haem and haemoglobin restored to almost normal the impaired growth rates of wild-type S. equi cultured under iron-limiting conditions. However, no difference in the growth rates of wild-type and mutants could be detected under the in vitro growth conditions tested.

Localization of the equine IgG-binding domain in the fibrinogen-binding protein (FgBP) of Streptococcus equi subsp. equi.

Fibrinogen-binding protein (FgBP, also termed SeM) is a cell-wall-associated anti-phagocytic M-like protein of the equine pathogen Streptococcus equi subsp. equi, and binds fibrinogen (Fg) and IgG. FgBP binds Fg avidly through residues located at the extreme N terminus of the molecule, whereas the IgG-binding site is more centrally located between the A and B repeats. FgBP binds equine IgG4 and IgG7 subclasses through interaction with the CH2-CH3 interdomain region of IgG-Fc, and possesses overlapping Fc-binding sites with protein A and protein G. In this study, FgBP truncates containing defined internal deletions were used to identify a stretch of 14 aa (residues 335-348) critical for IgG binding. Protein chimeras consisting of the non-IgG-binding alpha-helical coiled-coil M5 protein fused to FgBP sequences were used to identify a minimal equine IgG-binding domain consisting of residues 329-360. Competition ELISA tests suggested that IgG does not compromise Fg binding and vice versa.

Lack of correlation between antibody titers to fibrinogen-binding protein of Streptococcus equi and persistent carriers of strangles.

Previously published studies have neither used nor reported the results of an indirect enzyme-linked immunosorbent assay (iELISA) to measure serologic responses in natural outbreaks of strangles. The concept of using serologic responses to identify persistent carriers of Streptococcus equi has been proposed but not scientifically evaluated. The specific aims of the current study were to determine the duration and level of truncated fibrinogen-binding protein-specific (SeM allele 1) antibody production in ponies involved in a natural outbreak of strangles and to determine if test results from this serologic iELISA could predict persistent carrier status. Serologic samples were obtained before and after an outbreak of naturally occurring strangles infection. Persistent carriers of S. equi were identified via culture and polymerase chain reaction (PCR) testing of lavage fluid collected from the guttural pouches and nasopharynx or swabs of the nasopharynx after recovery from acute disease and at postmortem examination. Logistic regression analysis was used to determine if an association existed between serologic response and persistent carrier state. The ELISA reported in the current study definitively confirmed a recent exposure to S. equi. However, the measured serologic response did not predict carrier status in this strangles outbreak. Therefore, a guttural-pouch endoscopy with subsequent culture or PCR testing to detect S. equi remains the most accurate method available for the identification of persistent carriers.

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization.

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system.

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).

An investigation of criteria used to indicate cranial CT in males with schizophrenia.

BACKGROUND: With the increased accessibility of the CT scanner, psychiatrists managing schizophrenia and first-episode psychosis have incorporated this imaging technique into their diagnostic work-up. This practice has been reinforced by published criteria for CT scanning in psychiatric patients suggesting that cranial CT should be used as a screening tool to exclude intracranial pathology in all patients with a first presentation of schizophrenia or first psychotic episode. OBJECTIVES: This study reviews the performance of these criteria. METHOD: This consisted of a 3-year retrospective case-note audit, using published criteria, of all male in-patients with an established diagnosis of schizophrenia who had a cranial CT during the review period. RESULTS: The efficacy of the published criteria is not supported. In addition, non-specific abnormalities on cranial CT are related to duration of illness and not age in this sample. CONCLUSIONS: There is a need to establish new and clinically meaningful approaches for the use of cranial CT and similar technologies in clinical psychiatry. Cranial CT performs poorly as a universal screening tool in this population. The decision to use such imaging techniques should be made on a case-by-case basis using all available clinical evidence.

Competitive interaction of the OxyR DNA-binding protein and the Dam methylase at the antigen 43 gene regulatory region in Escherichia coli.

The antigen 43 surface protein of Escherichia coli is expressed in a phase-variable manner by a mechanism involving alternative activation and repression of transcription of the agn43 gene. The repressor is the OxyR DNA-binding protein, and its binding site was found to be located downstream of the agn43 transcription start site in a region of DNA that encompasses three 5'-GATC-3' sequences that are subject to Dam-mediated DNA methylation. It has been suggested previously that the phase-variable expression of antigen 43 results from a competition between Dam methylase and the OxyR repressor for these sites. The 5'-GATC-3' sequences were inactivated for methylation by site-directed mutagenesis, and all possible combinations of inactive and active sites were assessed for effects on phase-variable expression of the agn43 gene. Inactivation of any 5'-GATC-3' site individually had no effect; at least two sites had to be inactivated to disrupt the normal pattern of expression. Studies of OxyR interaction with agn43 DNA showed that methylation of any two 5'-GATC-3' sites was necessary and sufficient to block binding of the repressor. It was also found that the adenines of the second and third 5'-GATC-3' sites are required for OxyR binding, demonstrating that the sites for Dam methylation and for repressor binding are intimately associated. This is consistent with a competition model in which Dam and OxyR share a preference for specific DNA sequences in the regulatory region of the agn43 gene.

The C-terminal portion of the fibrinogen-binding protein of Streptococcus equi subsp. equi contains extensive alpha-helical coiled-coil structure and contributes to thermal stability.

The major cell wall-associated protein of the equine pathogen Streptococcus equi subsp. equi is a fibrinogen-binding protein (FgBP) which binds horse fibrinogen and equine IgG-Fc avidly through residues located in the N-terminal half and central regions of the molecule, respectively. The molecule is a major virulence factor for the organism and displays protective potential. In the present study, we use circular dichroism spectroscopy to investigate the secondary structure of the protein and show through the analysis of a panel of recombinant FgBP truncates that the C-terminal portion of FgBP contains an extensive alpha-helical coiled-coil structure that contributes to the thermal stability of the molecule.

Localization and characterization of the ligand-binding domain of the fibrinogen-binding protein (FgBP) of Streptococcus equi subsp. equi.

The group C streptococcus Streptococcus equi subsp. equi possesses a 498-residue major cell-wall-associated protein (FgBP) which binds horse fibrinogen (Fg), reacts with convalescent horse serum and protects against lethal S. equi challenge in a small animal model. In the present study, analysis of a panel of 17 purified N- and C-terminal FgBP truncates by ligand affinity blotting and SDS-PAGE revealed that the region required for maximum binding of Fg extended over the first half of the mature protein. The C-terminal two-thirds of this domain is predicted to be alpha-helical coiled-coil and the N-terminal one-third to possess non-coiled-coil single strands. Residues at the extreme N-terminus and within the coiled-coil region are both required for ligand binding. A high incidence of alpha-helical coiled-coil structure also seems to be responsible in part for the aberrant mobility of FgBP on SDS gels. The efficiency with which FgBP binds Fg from different animal species decreases in the order horse > mouse, pig > rat > sheep, dog, bovine, human. Binding to horse Fg is inversely related to temperature over the range 45-4 degrees C and is independent of Ca2+ ions. MS analysis provided corroborative evidence that FgBP is covalently linked to the cell wall peptidoglycan.

Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.

Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity.