Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture

A Case of Treatment Resistance and Complications in a Patient with Stiff Person Syndrome and Cerebellar Ataxia

BACKGROUND: Antibodies against glutamic acid decarboxylase (GAD) are associated with Stiff Person Syndrome (SPS). CASE REPORT: A 50-year-old woman presented with symptoms progressed over 9 years, resulting in a cerebellar ataxia and right upper limb tremor. Investigations revealed elevated serum and CSF anti-GAD antibody titres (98.6 and 53.4 μ/ml, respectively). Treatment included intravenous immunoglobulin and immunomodulation (infliximab and rituximab), improving her stiffness, but with no impact on the ataxia-related symptoms. Subsequent high-dose steroids led to diabetic ketoacidosis and unmasking of an insulin-dependent diabetes mellitus. DISCUSSION: This case illustrates several key features: (1) the combined clinical picture of SPS and cerebellar ataxia is a rare phenotype associated with anti-GAD antibodies; (2) the cerebellar ataxia described was progressive and poorly responsive to immunomodulatory therapy; and (3) the potential for development of further autoimmune sequelae in response to immunosuppression, namely, the development of insulin-dependent diabetes in response to treatment with high-dose oral steroids

Duration of antibiotic treatment for common infections in English primary care: cross sectional analysis and comparison with guidelines

Objectives: To evaluate antibiotic therapy durations for common infections in English primary care and to compare this with guidelines. Design: Cross-sectional study. Setting: General practices contributing to The Health Improvement Network database, 2013-2015. Participants: 931,015 consultations that resulted in an antibiotic prescription for one of the following indications: acute sinusitis, acute sore throat, acute cough and bronchitis, pneumonia, acute exacerbation of chronic obstructive pulmonary disease (COPD), acute otitis media, acute cystitis, prostatitis, pyelonephritis, cellulitis, impetigo, scarlet fever and gastroenteritis. Main outcome measures: The main outcomes were the percentage of antibiotic prescriptions with a duration exceeding the guideline recommendation and the total number of days beyond the recommended duration for each indication. Results: The most common reasons for the prescriptions were patients consulting with acute bronchitis and cough (386,972), acute sore throat (239,231), acute otitis media (83,054), and acute sinusitis (76,683). Antibiotic treatments for upper respiratory indications and acute bronchitis accounted for more than two thirds of the total prescriptions considered, and ≥80% of these treatment courses exceeded guideline recommendations. Notable exceptions were acute sinusitis, where only 9.6% (95% CI 9.4 to 9.9%) of prescriptions exceeded 7 days and acute sore throat where only 2.1% (95% CI 2.0 to 2.1) exceed 10 days (recent guidance recommends 5 days). More than half of antibiotic prescriptions were longer than guidelines recommend for acute cystitis among females (54.6%, 95% CI 54.1 to 55.0%). The percentage of antibiotic prescriptions exceeding the recommended duration was lower for most non-respiratory infections. For the 931,015 included consultations resulting in antibiotic prescriptions, approximately 1.3 million days were beyond the durations recommended by the guidelines. Conclusion: For most common infections treated in primary care, a substantial proportion of antibiotic prescriptions have durations exceeding those recommended in guidelines. Substantial reductions in antibiotic exposure can be accomplished by aligning antibiotic prescription durations with guidelines

Vector Positronium States in QED3

The homogeneous Bethe-Salpeter equation is solved in the quenched ladder approximation for the vector positronium states of 4-component quantum electrodynamics in 2 space and 1 time dimensions. Fermion propagator input is from a Rainbow approximation Dyson-Schwinger solution, with a broad range of fermion masses considered. This work is an extension of earlier work on the scalar spectrum of the same model. The non-relativistic limit is also considered via the large fermion mass limit. Classification of states via their transformation properties under discrete parity transformations allows analogies to be drawn with the meson spectrum of QCD.Comment: 24 pages, 2 encapsulated postscript figure

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1 dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces ICaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy

A national quality incentive scheme to reduce antibiotic overuse in hospitals; evaluation of perceptions and impact

In 2016/2017, a financially-linked antibiotic prescribing quality improvement initiative (AMR-CQUIN) was introduced across acute hospitals in England. This aimed for >1% reductions in Defined Daily Doses / 1000 admissions of total antibiotics, piperacillin/tazobactam and carbapenems compared with 2013/2014 and improved review of empiric antibiotic prescriptions

Intervention planning for Antibiotic Review Kit (ARK): a digital and behavioural intervention to safely review and reduce antibiotic prescriptions in acute and general medicine

Background Hospital antimicrobial stewardship strategies, such as ‘Start Smart, Then Focus’ in the UK, balance the need for prompt, effective antibiotic treatment with the need to limit antibiotic overuse using ‘review and revise’. However, only a minority of review decisions are to stop antibiotics. Research suggests that this is due to both behavioural and organizational factors. Objectives To develop and optimize the Antibiotic Review Kit (ARK) intervention. ARK is a complex digital, organizational and behavioural intervention that supports implementation of ‘review and revise’ to help healthcare professionals safely stop unnecessary antibiotics. Methods A theory-, evidence- and person-based approach was used to develop and optimize ARK and its implementation. This was done through iterative stakeholder consultation and in-depth qualitative research with doctors, nurses and pharmacists in UK hospitals. Barriers to and facilitators of the intervention and its implementation, and ways to address them, were identified and then used to inform the intervention’s development. Results A key barrier to stopping antibiotics was reportedly a lack of information about the original prescriber’s rationale for and their degree of certainty about the need for antibiotics. An integral component of ARK was the development and optimization of a Decision Aid and its implementation to increase transparency around initial prescribing decisions. Conclusions The key output of this research is a digital and behavioural intervention targeting important barriers to stopping antibiotics at review (see http://bsac-vle.com/ark-the-antibiotic-review-kit/ and http://antibioticreviewkit.org.uk/). ARK will be evaluated in a feasibility study and, if successful, a stepped-wedge cluster-randomized controlled trial at acute hospitals across the NHS

The antiphase boundary in half-metallic Heusler alloy Co2Fe(Al,Si) : atomic structure, spin polarization reversal, and domain wall effects

Atomic resolution scanning transmission electron microscopy reveals the presence of an antiphase boundary in the half-metallic Co2Fe(Al,Si) full Heusler alloy. By employing the density functional theory calculations, we show that this defect leads to reversal of the sign of the spin-polarization in the vicinity of the defect. In addition, we show that this defect reduces the strength of the exchange interactions, without changing the ferromagnetic ordering across the boundary. Atomistic spin calculations predict that this effect reduces the width of the magnetic domain wall compared to that in the bulk

Cost-effectiveness analysis of preoperative transfusion in patients with sickle cell disease using evidence from the TAPS trial.

The study's objective was to assess the cost-effectiveness of preoperative transfusion compared with no preoperative transfusion in patients with sickle cell disease undergoing low- or medium-risk surgery. Seventy patients with sickle cell disease (HbSS/Sß(0) thal genotypes) undergoing elective surgery participated in a multicentre randomised trial, Transfusion Alternatives Preoperatively in Sickle Cell Disease (TAPS). Here, a cost-effectiveness analysis based on evidence from that trial is presented. A decision-analytic model is used to incorporate long-term consequences of transfusions and acute chest syndrome. Costs and health benefits, expressed as quality-adjusted life years (QALYs), are reported from the 'within-trial' analysis and for the decision-analytic model. The probability of cost-effectiveness for each form of management is calculated taking into account the small sample size and other sources of uncertainty. In the range of scenarios considered in the analysis, preoperative transfusion was more effective, with the mean improvement in QALYs ranging from 0.018 to 0.206 per patient, and also less costly in all but one scenario, with the mean cost difference ranging from -£813 to £26. All scenarios suggested preoperative transfusion had a probability of cost-effectiveness >0.79 at a cost-effectiveness threshold of £20 000 per QALY