Multicenter Evaluation of the New Etest Gradient Diffusion Method for Piperacillin-Tazobactam Susceptibility Testing of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex.

Piperacillin-tazobactam (P/T) is a ß-lactam-ß-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales, P. aeruginosa, and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa, respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales, but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales, P. aeruginosa, and A. baumannii complex isolates with limited category errors.

Inhibition of Mycobacterium tuberculosis InhA: Design, synthesis and evaluation of new di-triclosan derivatives.

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. 'Direct inhibitors' of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC50 of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC99 of 12.9 µM against M. bovis.

Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa.

Ceftolozane-tazobactam (C/T) is a novel beta-lactam-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 Enterobacteriaceae challenge isolates and 39 Pseudomonas aeruginosa challenge isolates at three clinical sites. Essential agreement (EA) between the methods ranged from 47 to 49/51 (92.2 to 96.1%) for the Enterobacteriaceae, and categorical agreement (CA) ranged from 49 to 51/51 (96.1 to 100.0%). EA and CA for P. aeruginosa were 100% at all sites. The C/T Etest was also compared to BMD for susceptibility testing on 966 clinical isolates (793 Enterobacteriaceae, including 167 Klebsiella pneumoniae and 159 Escherichia coli isolates, in addition to 173 P. aeruginosa isolates) collected at four clinical sites. EA between Etest and BMD was 96.9% for Enterobacteriaceae isolates and 98.8% for P. aeruginosa isolates. Within the Enterobacteriaceae, isolates from each species examined had >96% CA. For the clinical isolates, no very major errors were identified but two major errors were found (one for K. pneumoniae and one for Providencia rettgeri). By BMD, 47.0% of Enterobacteriaceae and 46.2% of P. aeruginosa challenge strains were nonsusceptible to C/T by CLSI breakpoint criteria; 8.2% of clinical Enterobacteriaceae isolates and 12.1% of clinical P. aeruginosa isolates were nonsusceptible to C/T by CLSI breakpoint criteria. In conclusion, Etest is accurate and reproducible for C/T susceptibility testing of Enterobacteriaceae and P. aeruginosa.

Docking-Based Virtual Screening Enables Prioritizing Protein Kinase Inhibitors With In Vitro Phenotypic Activity Against Schistosoma mansoni.

Schistosomiasis is a parasitic neglected disease with praziquantel (PZQ) utilized as the main drug for treatment, despite its low effectiveness against early stages of the worm. To aid in the search for new drugs to tackle schistosomiasis, computer-aided drug design has been proved a helpful tool to enhance the search and initial identification of schistosomicidal compounds, allowing fast and cost-efficient progress in drug discovery. The combination of high-throughput in silico data followed by in vitro phenotypic screening assays allows the assessment of a vast library of compounds with the potential to inhibit a single or even several biological targets in a more time- and cost-saving manner. Here, we describe the molecular docking for in silico screening of predicted homology models of five protein kinases (JNK, p38, ERK1, ERK2, and FES) of Schistosoma mansoni against approximately 85,000 molecules from the Managed Chemical Compounds Collection (MCCC) of the University of Nottingham (UK). We selected 169 molecules predicted to bind to SmERK1, SmERK2, SmFES, SmJNK, and/or Smp38 for in vitro screening assays using schistosomula and adult worms. In total, 89 (52.6%) molecules were considered active in at least one of the assays. This approach shows a much higher efficiency when compared to using only traditional high-throughput in vitro screening assays, where initial positive hits are retrieved from testing thousands of molecules. Additionally, when we focused on compound promiscuity over selectivity, we were able to efficiently detect active compounds that are predicted to target all kinases at the same time. This approach reinforces the concept of polypharmacology aiming for "one drug-multiple targets". Moreover, at least 17 active compounds presented satisfactory drug-like properties score when compared to PZQ, which allows for optimization before further in vivo screening assays. In conclusion, our data support the use of computer-aided drug design methodologies in conjunction with high-throughput screening approach.

JMM Profile: Carbapenems: a broad-spectrum antibiotic.

Carbapenems are potent members of the ß-lactam family that inhibit bacterial cell-wall biosynthesis inhibitors . They are highly effective against Gram-negative and Gram-positive drug-resistant infections . As such, carbapenems are typically reserved as an antibiotic of last resort. The WHO lists meropenem as an essential medicine. Nausea and vomiting are reported in ≤20% of carbapenem recipients, with 1.5% suffering seizures. Enzymatic hydrolysis of the ß-lactam ring is the main driver of clinical resistance. These enzymes can be classified as Class A, B and D. Classes A and D are serine ß-lactamases, whereas Class B rely on metal-mediated hydrolysis, typically through zinc.

New InhA Inhibitors Based on Expanded Triclosan and Di-Triclosan Analogues to Develop a New Treatment for Tuberculosis.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new 'direct' InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expanding on the triclosan structure. Low Minimum Inhibitory Concentrations (MICs) were obtained for benzylphenyl compounds (12, 43 and 44) and di-triclosan derivative (39), against Mycobacterium bovis BCG although these may also be inhibiting other enzymes. The ether linked di-triclosan derivative (38) displayed excellent in vitro isolated enzyme inhibition results comparable with triclosan, but at a higher MIC (125 µg mL-1). These compounds offer good opportunities as leads for further optimisation.

Appendicitis with direct fistulation into the liver: a forgotten cause of pyogenic liver abscess.

Pyogenic liver abscess typically occurs secondary to biliary or haematogenous spread of organisms. In the context of acute appendicitis, abscesses generally occur due to haematogenous spread through the mesenteric vasculature. Historically, few cases of direct intra-abdominal spread have been reported but this has become vanishingly rare since the development of antibiotic therapy with no recorded cases in a search of over 900 cases in the literature.

Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors.

The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.

Differential habituation of fear and disgust during repeated exposure to threat-relevant stimuli in contamination-based OCD: an analogue study.

In the present study, participants (N=20) displaying marked contamination concerns were provided 30 min of repeated in vivo exposure to threat-relevant stimuli (cleaning a 'dirty' bed pan), during which time their fear and disgust levels were repeatedly assessed. Results indicated that repeated exposure led to a significant decline in fear but not disgust. The observed decline in fear remained significant after accounting for changes in disgust and vice versa. Although initial disgust was higher than initial fear ratings, differences between the two slopes were not statistically significant. Baseline trait anxiety and global disgust sensitivity levels prior to exposure did not moderate the level of fear or disgust activation during exposure. However, sensitivity specifically related to core and contamination disgust was marginally associated with fear and disgust parameters during outcome. There was also evidence that less fear decline during repeated exposure was associated with higher disgust ratings after the exposure was completed. Theoretical and clinical implications of the present findings for the treatment of contamination concerns in obsessive-compulsive disorder are discussed.

The instrumented instrument: characterization and training of manual skill in open suturing.

Instruction in the use of forceps and needle driver to suture tissues typically involves group didactic and demonstration sessions, followed by limited individual observation and coaching. Most motor learning required for dexterous suturing takes place during unsupervised practice with practice boards or during actual procedures in the operating room. We are developing surgical instruments with embedded microelectromechanical sensors for tracking instrument motion. Motion data is acquired and processed on a computer for concurrent or summary performance feedback during practice. An integral feature in our approach is the use of digital video recordings, synchronized with the sensor signals, to parse surgical procedures into a series of actions based on a task analysis. We envision the parsed video and signals as tools for assessment and performance feedback that an instructor could use to offer more extended individualized coaching. In a small pilot study, we concentrated our data analysis on the orientation of a needle driver about its long axis, the range of motion in one throw, and the timing of subtasks.

Authors' response: Letter to the Editor concerning OCRA as preferred method in ISO standards on biomechanical risk factors.

We thank Drs. Colombini and Occhipinti for their personal reply to our Discussion Paper (1, 2). We share the overall goal of preventing workplace injuries and welcome a discussion of the ISO process on workplace ergonomics standards; this was the primary aim of the Discussion Paper. We hope that other members of the relevant ISO working groups will also participate in the discussion. However, Drs. Colombini and Occipinti misinterpret our paper. Our aim was not to "addresses the scientific basis of ISO standards on biomechanical risk factors and more specifically the OCRA methodology". The purpose was to point out that "while the ISO process has value, it has also clear limitations when it comes to developing occupational health and safety standards that should be based on scientific principles". It is true that our paper discussed the OCRA method, but only as an example, in a single paragraph. We noted that the OCRA method was promoted as the preferred method by the ISO working group even though there were other risk assessment methods which, at the time (and currently), were at least as scientifically valid (3). The discovery that, while on the ISO working group, Drs. Colombini and Occipinti elevated the risk assessment method that they developed (OCRA) over the other methods, demonstrates one of several limitations of the ISO process, namely, the lack of attention to conflict of interest. Finally, we would like to draw attention to the note by Drs. Colombini and Occhipinti that "the ISO standards in question were actually developed by the working group, as mandated by ISO, over the period 2000‒2004". This long-elapsed time, without an update to the standard, should be a concern for all scientists given the large quantity of quality scientific literature published since then (eg, 3‒6). Fourteen years is well beyond what is recommended in the ISO guidelines. References 1. Colombini D, Occhipinti E. Scientific basis of the OCRA method for risk assessment of biomechanical overload of the upper limb, as preferred method in ISO standards on biomechanical risk factors. Scand J Work Environ Health ‒ online first. https://doi.org.10.5271/sjweh.3746 2. Armstrong T J, Burdorf I A, Descatha A, Farioli A, Graf M, Horie S, Marras W S, Potvin J R, Rempel D, Spatari G, Takala E P, Verbeek J, Violante FS. Scientific basis of ISO standards on biomechanical risk factors. Scand J Work Environ Health ‒ online first. https://doi.org/10.5271/sjweh.3718 3. Takala EP, Pehkonen I, Forsman M, Hansson GA, Mathiassen SE, Neumann WP, Sjøgaard G, Veiersted KB, Westgaard RH, Winkel J. Systematic evaluation of observational methods assessing biomechanical exposures at work. Scand J Work Environ Health. 2010;36:3-24. https://doi.org/10.5271/sjweh.2876 4. Paulsen R, Gallu T, Gilkey D, Reiser R, Murgia L, Rosecrance J. The inter-rater reliability of Strain Index and OCRA Checklist task assessments in cheese processing. Applied Ergonomics. 2015; 51,199-204. https://doi.org/10.1016/j.apergo.2015.04.019 5. Kapellusch JM, Gerr FE, Malloy EJ, Garg A, Harris-Adamson C, Bao SS, Burt SE, Dale AM, Eisen EA, Evanoff BA, Hegmann KT, Silverstein BA, Theise MS, Rempel DM. Exposure-response relationships for the ACGIH threshold limit value for hand-activity level: results from a pooled data study of carpal tunnel syndrome. Scand J Work Environ Health. 2014;40:610-20. https://doi.org/10.5271/sjweh.3456 6. Violante FS, Farioli A, Graziosi F, Marinelli F, Curti S, Armstrong TJ, Mattioli S, Bonfiglioli R. Carpal tunnel syndrome and manual work: the OCTOPUS cohort, results of a ten-year longitudinal study. Scand J Work Environ Health. 2016;42:280-90. https://doi.org/10.5271/sjweh.3566.

Scientific basis of ISO standards on biomechanical risk factors.

Among other purposes, companies and regulatory agencies from around the world often adopt International Standard Organization (ISO) standards to determine acceptable practices, equipment and criteria for preventing occupational injuries and illnesses. ISO standards are based on a consensus among individuals who participate in the process. This discussion paper examines the scientific process for the development of several ISO standards on biomechanical factors, comparing it with processes used by other professional organizations, including scientific committees working on the development of clinical guidelines. While the ISO process has value, it also has clear limitations when it comes to developing occupational health and safety standards that should be based on scientific principles.

PET-PANC: multicentre prospective diagnostic accuracy and health economic analysis study of the impact of combined modality 18fluorine-2-fluoro-2-deoxy-d-glucose positron emission tomography with computed tomography scanning in the diagnosis and management of pancreatic cancer.

BACKGROUND: Pancreatic cancer diagnosis and staging can be difficult in 10-20% of patients. Positron emission tomography (PET)/computed tomography (CT) adds precise anatomical localisation to functional data. The use of PET/CT may add further value to the diagnosis and staging of pancreatic cancer. OBJECTIVE: To determine the incremental diagnostic accuracy and impact of PET/CT in addition to standard diagnostic work-up in patients with suspected pancreatic cancer. DESIGN: A multicentre prospective diagnostic accuracy and clinical value study of PET/CT in suspected pancreatic malignancy. PARTICIPANTS: Patients with suspected pancreatic malignancy. INTERVENTIONS: All patients to undergo PET/CT following standard diagnostic work-up. MAIN OUTCOME MEASURES: The primary outcome was the incremental diagnostic value of PET/CT in addition to standard diagnostic work-up with multidetector computed tomography (MDCT). Secondary outcomes were (1) changes in patients' diagnosis, staging and management as a result of PET/CT; (2) changes in the costs and effectiveness of patient management as a result of PET/CT; (3) the incremental diagnostic value of PET/CT in chronic pancreatitis; (4) the identification of groups of patients who would benefit most from PET/CT; and (5) the incremental diagnostic value of PET/CT in other pancreatic tumours. RESULTS: Between 2011 and 2013, 589 patients with suspected pancreatic cancer underwent MDCT and PET/CT, with 550 patients having complete data and in-range PET/CT. Sensitivity and specificity for the diagnosis of pancreatic cancer were 88.5% and 70.6%, respectively, for MDCT and 92.7% and 75.8%, respectively, for PET/CT. The maximum standardised uptake value (SUVmax.) for a pancreatic cancer diagnosis was 7.5. PET/CT demonstrated a significant improvement in relative sensitivity (p = 0.01) and specificity (p = 0.023) compared with MDCT. Incremental likelihood ratios demonstrated that PET/CT significantly improved diagnostic accuracy in all scenarios (p < 0.0002). PET/CT correctly changed the staging of pancreatic cancer in 56 patients (p = 0.001). PET/CT influenced management in 250 (45%) patients. PET/CT stopped resection in 58 (20%) patients who were due to have surgery. The benefit of PET/CT was limited in patients with chronic pancreatitis or other pancreatic tumours. PET/CT was associated with a gain in quality-adjusted life-years of 0.0157 (95% confidence interval -0.0101 to 0.0430). In the base-case model PET/CT was seen to dominate MDCT alone and is thus highly likely to be cost-effective for the UK NHS. PET/CT was seen to be most cost-effective for the subgroup of patients with suspected pancreatic cancer who were thought to be resectable. CONCLUSION: PET/CT provided a significant incremental diagnostic benefit in the diagnosis of pancreatic cancer and significantly influenced the staging and management of patients. PET/CT had limited utility in chronic pancreatitis and other pancreatic tumours. PET/CT is likely to be cost-effective at current reimbursement rates for PET/CT to the UK NHS. This was not a randomised controlled trial and therefore we do not have any information from patients who would have undergone MDCT only for comparison. In addition, there were issues in estimating costs for PET/CT. Future work should evaluate the role of PET/CT in intraductal papillary mucinous neoplasm and prognosis and response to therapy in patients with pancreatic cancer. STUDY REGISTRATION: Current Controlled Trials ISRCTN73852054 and UKCRN 8166. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Fear and disgust processing during repeated exposure to threat-relevant stimuli in spider phobia.

Although disgust plays a significant role in the etiology of spider phobia, there remains a paucity of research examining the role of disgust in the treatment of spider phobia. Spider fearful participants (N = 46) were randomly assigned to a disgust (view vomit images) or neutral activation (view inanimate objects) condition. They were then repeatedly exposed to a videotaped tarantula, during which time their fear, disgust, and physiological levels were assessed repeatedly. Growth curve analyses indicated that repeated exposure led to significant declines in fear and disgust with no statistically significant differences between the two conditions. However, there was marginal evidence for decreased physiological arousal during repeated exposure among spider fearful participants in the disgust activation condition compared to those in the neutral condition. Reduction in disgust during exposure in the disgust activation condition remained significant after controlling for change in fear, whereas change in fear was no longer significant after controlling for change in disgust. However, the opposite pattern of relations between change in fear and disgust was observed in the neutral activation condition. Higher fear and disgust activation during exposure was also associated with higher fear and disgust responding on a subsequent behavioral task and higher spider fear and disgust at 3-month follow-up. Baseline trait disgust propensity also predicted fear and disgust parameters during repeated exposure. The implications of these findings for the role of disgust in the treatment of spider phobia are discussed.