Performance of a System for Rapid Phenotypic Antimicrobial Susceptibility Testing of Gram-Negative Bacteria Directly from Positive Blood Culture Bottles.

The rapid administration of optimal antimicrobial treatment is paramount for the treatment of bloodstream infections (BSIs), and rapid antimicrobial susceptibility testing (AST) results are essential. Q-linea has developed the ASTar system, a rapid phenotypic AST device. Here, we report the performance of the ASTar BC G- (Gram-negative) kit when assessed according to the ISO 20776-2:2007 standard for performance evaluation of in vitro diagnostic AST devices. The evaluated ASTar BC G- kit uses a broad panel of 23 antimicrobials for the treatment of BSIs caused by Gram-negative fastidious and nonfastidious bacteria across a range of 6 to 14 2-fold dilutions, including cefoxitin as a screening agent for AmpC-producing Enterobacterales. The ASTar system processes blood culture samples to generate data on MICs and susceptible, intermediate, or resistant (SIR) category. The automated protocol includes concentration determination and concentration adjustment to enable a controlled inoculum, followed by broth microdilution (BMD) and microscopy performed continuously to generate MIC values within approximately 6 h once the test is run on the ASTar system. The performance of the ASTar system was assessed against the ISO 20776-2:2007 standard BMD reference method. Testing was performed across three sites, with results from 412 contrived blood cultures and 74 fresh clinical blood cultures. The ASTar system was also tested for reproducibility, with triplicate testing of 11 strains. The accuracy study comprised 8,650 data points of bacterium-antimicrobial tests. The ASTar system demonstrated an overall essential agreement (EA) of 95.8% (8,283/8,650) and a categorical agreement (CA) of 97.6% (8,433/8,639) compared to the reference BMD method. The overall rate of major discrepancies (MDs) was 0.9% (62/6,845), and that of very major discrepancies (VMDs) was 2.4% (30/1,239). This study shows that the ASTar system delivers reproducible results with overall EA and CA of >95%.

The value of a new cardiac magnetic resonance imaging protocol in Myocardial Infarction with Non-obstructive Coronary Arteries (MINOCA) - a case-control study using historical controls from a previous study with similar inclusion criteria.

BACKGROUND: Myocardial Infarction with Non-Obstructive Coronary Arteries (MINOCA) is common with a prevalence of 6% of all patients fulfilling the diagnosis of myocardial infarction. MINOCA should be considered a working diagnosis. Cardiac Magnetic Resonance (CMR) imaging has recently been suggested to be of great value to determine the cause behind MINOCA. The objectives of this paper are to describe the rationale behind the second Stockholm Myocardial Infarction with Normal Coronaries (SMINC-2) study and to discuss the protocol for investigation of MINOCA patients in the light of the recently published position paper from the European Society of Cardiology. METHODS: The SMINC-2 study is an open non-randomised study using historical controls for comparison. The primary aim is to prove that MINOCA patients investigated with the latest CMR imaging technique can achieve a diagnosis in 70% of all cases entirely by imaging. By including 150 patients we will have >80% chance to prove that the diagnostic accuracy can be improved by 20 absolute % with a p-value of less than 0.05 when compared with CMR imaging in the SMINC-1 study. Furthermore, in addition to invasive coronary angiography, coronary arteries are evaluated by computed tomography angiography to investigate coronary causes and questionnaires are used to describe Quality-of-Life (QoL). By January 1st 2017, 75 patients have been included. DISCUSSION: Whether CMR imaging can provide a diagnosis to an adequate proportion of MINOCA patients is unknown. Well-defined inclusion and exclusion criteria will be used to compare a MINOCA cohort from the population with an appropriate control group. Positive results are likely to influence future guidelines of the management of MINOCA. Furthermore, the study will give mechanistic insights into MINOCA in particular in patients with "true" myocardial infarction and describe QoL in this vulnerable group of patients. TRIAL REGISTRATION: Clinical Trials NCT02318498 .

Glucosinolate-rich broccoli sprouts protect against oxidative stress and improve adaptations to intense exercise training.

Oxidative stress plays a vital role for the adaptive responses to physical training. However, excessive oxidative stress can precipitate cellular damage, necessitating protective mechanisms to mitigate this effect. Glucosinolates, found predominantly in cruciferous vegetables, can be converted into isothiocyanates, known for their antioxidative properties. These compounds activate crucial antioxidant defence pathways and support mitochondrial function and protein integrity under oxidative stress, in both Nrf2-dependent and independent manners. We here administered glucosinolate-rich broccoli sprouts (GRS), in a randomized double-blinded cross-over fashion to 9 healthy subjects in combination with daily intense exercise training for 7 days. We found that exercise in combination with GRS significantly decreased the levels of carbonylated proteins in skeletal muscle and the release of myeloperoxidase into blood. Moreover, it lowered lactate accumulation during submaximal exercise, and attenuated the severe nocturnal hypoglycaemic episodes seen during the placebo condition. Furthermore, GRS in combination with exercise improved physical performance, which was unchanged in the placebo condition.

Little evidence for reversibility of trimethoprim resistance after a drastic reduction in trimethoprim use.

OBJECTIVES: The worldwide rapid increase in antibiotic-resistant bacteria has made efforts to prolong the lifespan of existing antibiotics very important. Antibiotic resistance often confers a fitness cost in the bacterium. Resistance may thus be reversible if antibiotic use is discontinued or reduced. To examine this concept, we performed a 24 month voluntary restriction on the use of trimethoprim-containing drugs in Kronoberg County, Sweden. METHODS: The intervention was performed on a 14 year baseline of monthly data on trimethoprim resistance and consumption. A three-parameter mathematical model was used to analyse the intervention effect. The prerequisites for reversion of resistance (i.e. fitness cost, associated resistance and clonal composition) were studied on subsets of consecutively collected Escherichia coli from urinary tract infections. RESULTS: The use of trimethoprim-containing drugs decreased by 85% during the intervention. A marginal but statistically significant effect on the increase in trimethoprim resistance was registered. There was no change in the clonal composition of E. coli and there was no measurable fitness cost associated with trimethoprim resistance in clinical isolates. The frequency of associated antibiotic resistances in trimethoprim-resistant isolates was high. CONCLUSIONS: A lack of detectable fitness cost of trimethoprim resistance in vitro together with a strong co-selection of other antibiotics could explain the rather disappointing effect of the intervention. The result emphasizes the low possibility of reverting antibiotic resistance once established and the urgent need for the development of new antibacterial agents.

Evaluation of the FilmArray™ Meningitis/Encephalitis panel with focus on bacteria and Cryptococcus spp.

PURPOSE: Molecular methods provide fast and accurate detection of both bacteria and viruses in the cerebrospinal fluid (CSF) causing infection in the central nervous system (CNS). In the present study we evaluated the bacterial detection performance of the fully automated FilmArray™ Meningitis/Encephalitis (ME) panel (bioMérieux) by comparing it with culture and multiplexed in-house PCR. METHODS: Three sample types were analysed; Contrived samples with known bacterial/fungal concentration (n = 29), clinical samples from patients with verified cause of CNS infection (n = 17) and external quality assessment (EQA) samples (n = 11). Another six samples were purposely prepared with multiple targets to evaluate multiplex capacity. RESULTS: The FilmArray™ had a slightly higher limit of detection for Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes and Streptococcus agalactiae compared to in-house PCR methods but performed equal or better when compared to culture. The FilmArray™ ME panel detected the expected pathogen in 17 of 17 clinical samples and yielded detection of three additional viruses of which one was confirmed with comparator techniques. All but one of the EQA samples were correctly detected. CONCLUSIONS: The results of this study are promising and the FilmArray™ ME panel could add to the diagnostic algorithm in CNS-infections. However, the limit of detection for the important pathogens N. meningitidis and S. pneumoniae could be improved.

Formyl peptide receptor 2 orchestrates mucosal protection against Citrobacter rodentium infection.

Citrobacter rodentium is an attaching and effacing intestinal murine pathogen which shares similar virulence strategies with the human pathogens enteropathogenic- and enterohemorrhagic Escherichia coli to infect their host. C. rodentium is spontaneously cleared by healthy wild-type (WT) mice whereas mice lacking Muc2 or specific immune regulatory genes demonstrate an impaired ability to combat the pathogen. Here we demonstrate that apical formyl peptide receptor 2 (Fpr2) expression increases in colonic epithelial cells during C. rodentium infection. Using a conventional inoculum dose of C. rodentium, both WT and Fpr2-/- mice were infected and displayed similar signs of disease, although Fpr2-/- mice recovered more slowly than WT mice. However, Fpr2-/- mice exhibited increased susceptibility to C. rodentium colonization in response to low dose infection: 100% of the Fpr2-/- and 30% of the WT mice became colonized and Fpr2-/- mice developed more severe colitis and more C. rodentium were in contact with the colonic epithelial cells. In line with the larger amount of C. rodentium detected in the spleen in Fpr2-/- mice, more C. rodentium and enteropathogenic Escherichia coli translocated across an in vitro mucosal surface to the basolateral compartment following FPR2 inhibitor treatment. Fpr2-/- mice also lacked the striated inner mucus layer that was present in WT mice. Fpr2-/- mice had decreased mucus production and different mucin O-glycosylation in the colon compared to WT mice, which may contribute to their defect inner mucus layer. Thus, Fpr2 contributes to protection against infection and influence mucus production, secretion and organization.

Microbiological diagnostics of bloodstream infections in Europe-an ESGBIES survey.

OBJECTIVES: High-quality diagnosis of bloodstream infections (BSI) is important for successful patient management. As knowledge on current practices of microbiological BSI diagnostics is limited, this project aimed to assess its current state in European microbiological laboratories. METHODS: We performed an online questionnaire-based cross-sectional survey comprising 34 questions on practices of microbiological BSI diagnostics. The ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES) was the primary platform to engage national coordinators who recruited laboratories within their countries. RESULTS: Responses were received from 209 laboratories in 25 European countries. Although 32.5% (68/209) of laboratories only used the classical processing of positive blood cultures (BC), two-thirds applied rapid technologies. Of laboratories that provided data, 42.2% (78/185) were able to start incubating BC in automated BC incubators around-the-clock, and only 13% (25/192) had established a 24-h service to start immediate processing of positive BC. Only 4.7% (9/190) of laboratories validated and transmitted the results of identification and antimicrobial susceptibility testing (AST) of BC pathogens to clinicians 24 h/day. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from briefly incubated sub-cultures on solid media was the most commonly used approach to rapid pathogen identification from positive BC, and direct disc diffusion was the most common rapid AST method from positive BC. CONCLUSIONS: Laboratories have started to implement novel technologies for rapid identification and AST for positive BC. However, progress is severely compromised by limited operating hours such that current practice of BC diagnostics in Europe complies only partly with the requirements for optimal BSI management.

Clinical and analytical evaluation of the new Aptima Mycoplasma genitalium assay, with data on M. genitalium prevalence and antimicrobial resistance in M. genitalium in Denmark, Norway and Sweden in 2016.

OBJECTIVES: Mycoplasma genitalium (MG) causes urethritis and cervicitis, potentially causing reproductive complications. Resistance in MG to first-line (azithromycin) and second-line (moxifloxacin) treatment has increased. We examined the clinical and analytical performance of the new Conformité Européene (CE)/in vitro diagnostics (IVD) Aptima Mycoplasma genitalium assay (CE/IVD AMG; Hologic); the prevalence of MG, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG); and MG resistance to azithromycin and moxifloxacin in Denmark, Norway and Sweden in 2016. METHODS: From February 2016 to February 2017, urogenital and extragenital (only in Denmark) specimens from consecutive attendees at three sexually transmitted disease clinics were tested with the CE/IVD AMG, the research-use-only MG Alt TMA-1 assay (Hologic), Aptima Combo 2 (CT/NG) assay and a laboratory-developed TaqMan real-time mgpB quantitative real-time PCR (qPCR). Resistance-associated mutations were determined by sequencing. Strains of MG and other mycoplasma species in different concentrations were also tested. RESULTS: In total 5269 patients were included. The prevalence of MG was 7.2% (382/5269; 4.9-9.8% in the countries). The sensitivity of the CE/IVD AMG, MG Alt TMA-1 and mgpB qPCR ranged 99.13-100%, 99.13-100% and 73.24-81.60%, respectively, in the countries. The specificity ranged 99.57-99.96%, 100% and 99.69-100%, respectively. The prevalence of resistance-associated mutations for azithromycin and moxifloxacin was 41.4% (120/290; 17.7-56.6%) and 6.6% (18/274; 4.1-10.2%), respectively. Multidrug resistance was found in all countries (2.7%; 1.1-4.2%). CONCLUSIONS: Both transcription-mediated amplification (TMA)-based MG assays had a highly superior sensitivity compared to the mgpB qPCR. The prevalence of MG and azithromycin resistance was high. Validated and quality-assured molecular tests for MG, routine resistance testing of MG-positive samples and antimicrobial resistance surveillance are crucial.

The aetiology of pharyngotonsillitis in adolescents and adults - Fusobacterium necrophorum is commonly found.

Sore throat is common in primary healthcare. Aetiological studies have focused on the presence of a limited number of pathogens. The aim of the present study was to investigate the presence of a wide range of bacteria and viruses, including Fusobacterium necrophorum, in patients with pharyngotonsillitis and in asymptomatic controls. A prospective case control study was performed in primary healthcare in Kronoberg County, Sweden. Patients (n=220) aged 15 to 45 years with a suspected acute pharyngotonsillitis, and controls (n=128), were included. Nasopharyngeal and throat swabs were analysed for ß-hemolytic streptococci, F. necrophorum, Mycoplasma pneumoniae, and Chlamydophila pneumoniae, and 13 respiratory viruses. Serum samples were analysed for antibodies to Epstein-Barr virus. The patient history and symptoms, including Centor score, were analysed in relation to pathogens. In 155/220 (70.5%) of the patients, as compared to 26/128 (20.3%) of the controls (p <0.001), at least one microorganism was found. Group A streptococci, F. necrophorum, and influenza B virus were the three most common findings, and all significantly more common in patients than in controls (p <0.001, p 0.001, and p 0.002, respectively). Patients with F. necrophorum only (n=14) displayed a lower Centor score than patients with Group A streptococcus only (n=46), but a higher score than patients with influenza B, other viruses, or no potential pathogen (Kruskal-Wallis p <0.001). A pathogen was detected in 70% of the patients, displaying a wide range of pathogens contributing to the aetiology of pharyngotonsillitis. This study supports F. necrophorum as one of the pathogens to be considered in the aetiology of pharyngotonsillitis.

Quantifying and Communicating Uncertainty in Preclinical Human Dose-Prediction.

Human dose-prediction is fundamental for ranking lead-optimization compounds in drug discovery and to inform design of early clinical trials. This tutorial describes how uncertainty in such predictions can be quantified and efficiently communicated to facilitate decision-making. Using three drug-discovery case studies, we show how several uncertain pieces of input information can be integrated into one single uncomplicated plot with key predictions, including their uncertainties, for many compounds or for many scenarios, or both.

Prolonged tamoxifen treatment increases relapse-free survival for patients with primary breast cancer expressing high levels of VEGF.

Previous retrospective studies have shown that high intratumoural levels of vascular endothelial growth factor (VEGF) correlate with an inferior outcome for patients treated with adjuvant tamoxifen. Our objectives were to validate the impact of VEGF on survival after adjuvant tamoxifen and to investigate the interaction between VEGF and treatment duration. For this purpose tumour homogenates from 402 patients with operable oestrogen receptor positive breast cancer (BC), treated with tamoxifen for 2 (n=149) or 5 years (n=253) as the only systemic adjuvant therapy were included. The median follow-up time for surviving patients was 9.8 years (range 0.5-14.8 years). Expression of VEGF was assessed by an enzyme-linked immunosorbent assay and investigated in relation to the standard BC parameters and survival. In the total population, higher VEGF was significantly correlated with shorter recurrence-free survival (RFS) (HR=1.63, 95%CI=1.11-2.39, p=0.010), breast cancer corrected survival (BCCS) (HR=1.82, 95%CI=1.13-2.93, p=0.014) and overall survival (OS) (HR=1.51, 95%CI=1.11-2.05, p=0.009). High VEGF was significantly associated with reduced RFS (HR=2.61, 95%CI=1.45-4.70, p=0.001) after two years of tamoxifen, whilst no difference was seen in patients treated for five years (HR=1.09, 95%CI=0.64-1.84, p=0.760). A statistically significant interaction was observed between high VEGF expression and improved RFS after 5-year tamoxifen (p=0.034). In concordance with previous studies, high VEGF was significantly correlated with shorter survival. We present data not reported previously revealing that patients expressing high levels of VEGF display a better outcome provided that tamoxifen is given for five years. Further studies on the impact of VEGF on a 5-year regimen are motivated.

Fexofenadine in horses: pharmacokinetics, pharmacodynamics and effect of ivermectin pretreatment.

The pharmacokinetics and the effects on inhibition of histamine-induced cutaneous wheal formation of the histamine H1-antagonist fexofenadine were studied in horse. The effect of ivermectin pretreatment on the pharmacokinetics of fexofenadine was also examined. After intravenous infusion of fexofenadine at 0.7 mg/kg bw the mean terminal half-life was 2.4 h (range: 2.0-2.7 h), the apparent volume of distribution 0.8 L/kg (0.5-0.9 L/kg), and the total body clearance 0.8 L/h/kg (0.6-1.2 L/h/kg). After oral administration of fexofenadine at 10 mg/kg bw bioavailability was 2.6% (1.9-2.9%). Ivermectin pretreatment (0.2 mg/kg, p.o.) 12 h before oral fexofenadine decreased the bioavailability to 1.5% (1.4-2.1%). In addition, the area under the plasma concentration-time curve decreased 27%. Ivermectin did not affect the pharmacokinetics of i.v. administered fexofenadine. Ivermectin may influence fexofenadine absorption by interfering in intestinal efflux and influx pumps, such as P-glycoprotein and the organic anion transport polypeptide family. Oral and i.v. fexofenadine significantly decreased histamine-induced wheal formation, with a maximal duration of 6 h. A pharmacokinetic/pharmacodynamic link model indicated that fexofenadine in horse has antihistaminic effects at low plasma concentrations (EC50 = 16 ng/mL). However, oral treatments of horses with fexofenadine may not be suitable due to the low bioavailability.

Circulating ghrelin levels after food intake during different phases of the migrating motor complex in man.

BACKGROUND: The timing of the migrating motor complexes (MMC) at food intake may influence gastric emptying and release of regulatory hormones. This report studies the relationships between phases I (motor quiescence) and II (intermediate frequency contractions) of MMC and prandial gut hormone response. MATERIALS AND METHODS: Seven fasting volunteers ingested a meal during phase I or II of MMC verified by manometry, using paracetamol as a marker for gastric emptying. Blood was sampled before, during and 210 min after food intake for analysis of ghrelin, motilin, insulin and paracetamol. RESULTS: The basal level of ghrelin during phase I was 127.5 +/- 25.4 pmol L(-1) and during phase II was 132.4 +/- 24.8 pmol L(-1). After food intake during phase I, ghrelin fell to 77.2 +/- 10 pmol L(-1); in phase II it fell to 82.7 +/- 17.8 pmol L(-1) within 60 min and returned to baseline levels after 120 min. Baseline levels of motilin were 16 +/- 2 pmol L(-1) and 18 +/- 3 pmol L(-1) during phases I and II, respectively. After food, motilin decreased to 8.5 +/- 0.7 pmol L(-1) and 8.7 +/- 1.0 pmol L(-1) within 60 min and returned to baseline after 90 min. Insulin levels in phases I and II were 8.1 +/- 1.2 mU L(-1) and 8.6 +/- 0.7 mU L(-1), respectively, reaching 138.9 +/- 35.6 mU L(-1) and 167.4 +/- 30.0 mU L(-1) at 45 min postprandially. CONCLUSIONS: The nutritional status of the gastrointestinal tract at food intake had only a limited impact on plasma ghrelin. After food intake, plasma ghrelin drops, similar to motilin, and resumes preprandial levels within 120 min.

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian Xenopus laevis.

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57-66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61-62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.