Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively

Why pharmacokinetic differences among oral triptans have little clinical importance: a comment

Triptans, selective 5-HT1B/1D receptor agonists, are specific drugs for the acute treatment of migraine that have the same mechanism of action. Here, it is discussed why the differences among kinetic parameters of oral triptans have proved not to be very important in clinical practice. There are three main reasons: (1) the differences among the kinetic parameters of oral triptans are smaller than what appears from their average values; (2) there is a large inter-subject, gender-dependent, and intra-subject (outside/during the attack) variability of kinetic parameters related to the rate and extent of absorption, i.e., those which are considered as critical for the response; (3) no dose-concentration–response curves have been defined and it is, therefore, impossible both to compare the kinetics of triptans, and to verify the objective importance of kinetic differences; (4) the importance of kinetic differences is outweighed by non-kinetic factors of variability of response to triptans. If no oral formulations are found that can allow more predictable pharmacokinetics, the same problems will probably also arise with new classes of drugs for the acute treatment of migraine

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The outcome of initiation of antiepileptic drug monotherapy in primary care: a UK database survey

We describe the incidence of newly treated epilepsy in primary care and patterns of antiepileptic drug prescription, numbers of patients who remain on initial therapy and health service utilisation. Data was collected from 100 general practices that subscribed to the Doctors Independent Network (DIN-LINK) project. Over the study period 1531 patients were identified, equating to an annual incidence rate of 36.3 per 100 000 (95% confidence interval [CI] = 32.1 to 40.8). Of these patients, 1465 (95.7%) were started on antiepileptic drugs. Overall, 1154 (78.8%) patients remained on the original monotherapy at the 12-month stage. Primary care consultations, secondary care referrals and emergency admissions were all increased for those whose treatment was changed either to polytherapy or an alternative monotherapy