Rapid measurement of tacrolimus in whole blood by paper spray-tandem mass spectrometry (PS-MS/MS)

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides sensitivity and specificity for monitoring tacrolimus drug level in blood, but it requires an LC system and sample preparation, which is not amenable to random access testing typical of immunoassays. Paper spray (PS) ionization generates gas phase analyte ions directly from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS method for tacrolimus drug monitoring in a clinical diagnostic laboratory. Methods Whole blood sample was mixed with stable isotope labeled internal standard ([13C, 2H2]-FK506) and spotted onto a cartridge containing triangular shaped card paper. After drying, samples were analyzed by PS MS/MS in the selected reaction monitoring (SRM) mode, with a run time of 3 min/sample. Results Analytical measurement range was 1.5–30 ng/ml. Assay inter-day imprecision was 13%, 8%, and 5% at tacrolimus concentrations of 4.5, 10.5, and 24.5 ng/ml, respectively. Accuracy was determined by pure tacrolimus solution and was confirmed by result correlation to an immunoassay (slope = 1.0, intercept = − 0.02; r2 = 0.99), and to a conventional LC-MS/MS method (slope = 0.90, intercept = 0.4; r2 = 0.94). Conclusions PS-MS/MS provides accurate results for tacrolimus with rapid turnaround time amenable to random access testing protocols

A retrospective observational study of traumatic orthopaedic: related infections in Cambodia

Background: The objective of this study was to establish the type of microbiology along with antimicrobial resistance related to orthopedic related trauma infections in this area in order to help guide diagnosis and treatment regimens.Methods:This study evaluated the microbial etiology of orthopedic-related infections (ORI) between September 2015 and September 2016 in three tertiary hospitals in Phnom Penh, Cambodia. Clinical records were for clinical features and demographics. Standard laboratory bacteriology was used to recover, identified and perform antibiotic susceptibility testing (AST) by disk diffusion or broth microdilution.Results:119 patients were categorized as ORI cases. In the cases identified, median interquartile range (IQR) age was 38 (IQR: 26-46) years and 80.0% were male. Of the 119 ORI cases, a total of 156 bacterial strains were recovered, identified and after review, 128 of these pathogenic bacterial strains underwent AST. Among the gram-positive pathogens, the following susceptibilities were as follows: Staphylococcus aureus (n=57) (Methicillin-resistant S. aureus (n=35; 61.4%), (Methicillin‐sensitive S. aureus (n=22; 38.6%)), coagulase-negative staphylococcus (all MS-CoNS; n=6) and four isolates of Enterococcus sp. (non-VRE). A total of 44 gram-negative pathogens were recovered and AST was performed. Among these 44, a total of nine extended-spectrum beta-lactamase (ESBL) producing strains (20.5%) were discovered including Escherichia coli (n=8), Klebsiella pneumoniae (n=1) and carbapenemase-resistant Enterobacteriaceae (CRE) (Morganella morganii). In addition, a single E. coli isolate contained both the ESBL and CRE genotypes was noted.Conclusions:This data suggests that ORI rates in Cambodia appear to be comparable to other studies in the literature. However, further studies need to be done in order to establish definitive data related to orthopedic infections in the region

A progress report of the IFCC Committee for Standardization of Thyroid Function Tests

BACKGROUND: The IFCC Committee for Standardization of Thyroid Function Tests aims at equivalence of laboratory test results for free thyroxine (FT4) and thyrotropin (TSH). OBJECTIVES: This report describes the phase III method comparison study with clinical samples representing a broad spectrum of thyroid disease. The objective was to expand the feasibility work and explore the impact of standardization/harmonization in the clinically relevant concentration range. METHODS: Two sets of serum samples (74 for FT4, 94 for TSH) were obtained in a clinical setting. Eight manufacturers participated in the study (with 13 FT4 and 14 TSH assays). Targets for FT4 were set by the international conventional reference measurement procedure of the IFCC; those for TSH were based on the all-procedure trimmed mean. The manufacturers recalibrated their assays against these targets. RESULTS: All FT4 assays were negatively biased in the mid- to high concentration range, with a maximum interassay discrepancy of approximately 30%. However, in the low range, the maximum deviation was approximately 90%. For TSH, interassay comparability was reasonable in the mid-concentration range, but worse in the pathophysiological ranges. Recalibration was able to eliminate the interassay differences, so that the remaining dispersion of the data was nearly entirely due to within-assay random error components. The impact of recalibration on the numerical results was particularly high for FT4. CONCLUSIONS: Standardization and harmonization of FT4 and TSH measurements is feasible from a technical point of view. Because of the impact on the numerical values, the implementation needs careful preparation with the stakeholders

Triple Combination of Amantadine, Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza

Early blood pressure, antihypotensive therapy and outcomes at 18–22 months’ corrected age in extremely preterm infants

Investigate relationships between early blood pressure (BP) changes, receipt of anti-hypotensive therapy, and 18 – 22 month corrected age (CA) outcomes for extremely preterm infants

A whole genome RNAi screen of Drosophila S2 cell spreading performed using automated computational image analysis

An automated, image-based RNAi screen for cell shape reveals roles for membrane secretion factors in cell spreading

Using Single loxP Sites to Enhance Homologous Recombination: ts Mutants in Sec1 of Dictyostelium discoideum

Dictyostelium discoideum amoebae are haploid and, as they share many features with animal cells, should be an ideal creature for studying basic processes such as cell locomotion. Isolation of mutants in this amoeba has largely been limited to non-essential genes: nsfA-the gene for NEM-sensitive factor-remains the only essential gene for which conditional (ts) mutants exist. These ts mutants were generated by gene replacement using a library of mutagenised nsfA containing a selectable marker: transformants were then screened for temperature sensitivity. The success of this approach depended on the high level of homologous recombination prevailing at this locus: approximately 95% of selected clones were homologous recombinants. This is unusually high for Dictyostelium: homologous recombination at other loci is usually much less, usually between 0-30%, making the isolation of ts mutants much more tedious.In trying to make ts mutants in sec1A, homologous recombination was found to be only approximately 25%. A new approach, involving single loxP sites, was investigated. LoxP sites are 34 bp sequences recognised by Cre recombinase and between which this enzyme catalyses recombination. A Dictyostelium line containing a single loxP site adjacent to the 3' end of the sec1A gene was engineered. A sec1A replacement DNA also containing a single loxP site in a homologous position was then introduced into this cell line. In the presence of CRE recombinase, homologous recombination increased to approximately 80% at this locus, presumably largely driven by intermolecular recombination between the two single loxP sites.A route to increase the rate of homologous recombination at a specific locus, sec1A, is described which enabled the isolation of 30 ts mutants in sec1A. One of these, sec1Ats1,has been studied and found to cease moving at the restrictive temperature. The approach described here may be valuable for enhancing homologous recombination at specified loci and thus for introducing mutations into specific genes in Dictyostelium and other creatures

Regulation of the Actin Cytoskeleton by an Interaction of IQGAP Related Protein GAPA with Filamin and Cortexillin I

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin

Persistence of serogroup C antibody responses following quadrivalent meningococcal conjugate vaccination in United States military personnel

AbstractSerogroup C meningococcal (MenC) disease accounts for one-third of all meningococcal cases and causes meningococcal outbreaks in the U.S. Quadrivalent meningococcal vaccine conjugated to diphtheria toxoid (MenACYWD) was recommended in 2005 for adolescents and high risk groups such as military recruits. We evaluated anti-MenC antibody persistence in U.S. military personnel vaccinated with either MenACYWD or meningococcal polysaccharide vaccine (MPSV4). Twelve hundred subjects vaccinated with MenACYWD from 2006 to 2008 or MPSV4 from 2002 to 2004 were randomly selected from the Defense Medical Surveillance System. Baseline serologic responses to MenC were assessed in all subjects; 100 subjects per vaccine group were tested during one of the following six post-vaccination time-points: 5–7, 11–13, 17–19, 23–25, 29–31, or 35–37 months. Anti-MenC geometric mean titers (GMT) were measured by rabbit complement serum bactericidal assay (rSBA) and geometric mean concentrations (GMC) by enzyme-linked immunosorbent assay (ELISA). Continuous variables were compared using the Wilcoxon rank sum test and the proportion of subjects with an rSBA titer ≥8 by chi-square. Pre-vaccination rSBA GMT was <8 for the MenACWYD group. rSBA GMT increased to 703 at 5–7 months post-vaccination and decreased by 94% to 43 at 3 years post-vaccination. GMT was significantly lower in the MenACWYD group at 5–7 months post-vaccination compared to the MPSV4 group. The percentage of MenACWYD recipients achieving an rSBA titer of ≥8 decreased from 87% at 5–7 months to 54% at 3 years. There were no significant differences between vaccine groups in the proportion of subjects with a titer of ≥8 at any time-point. GMC for the MenACWYD group was 0.14μg/mL at baseline, 1.07μg/mL at 5–7 months, and 0.66μg/mL at 3 years, and significantly lower than the MPSV4 group at all time-points. Anti-MenC responses wane following vaccination with MenACYWD; a booster dose is needed to maintain protective levels of circulating antibody