Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection.

BACKGROUND: The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood. METHODS: Blood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol-SepsiPURE. RESULTS: After first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50-100 colony forming units (CFU)/mL to 1-5 CFU/mL. CONCLUSION: In this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI.

Rapid Rule Out of Culture-Negative Bloodstream Infections by Use of a Novel Approach to Universal Detection of Bacteria and Fungi.

BACKGROUND: Currently it can take up to 5 days to rule out bloodstream infection. With the low yield of blood cultures (approximately 10%), a significant number of patients are potentially exposed to inappropriate therapy that can lead to adverse events. More rapid rule out can accelerate deescalation or cessation of antimicrobial therapy, improving patient outcomes. METHODS: A method is described, termed enzymatic template generation and amplification (ETGA), that universally and sensitively detects DNA polymerase activity liberated from viable bacteria and fungi isolated from blood culture samples as a measure of bloodstream infection. ETGA was applied in a diagnostic test format to identify negative blood cultures after an overnight incubation. Performance data for a prototype (Cognitor) and automated (Magnitor) version of the test are presented. RESULTS: The Cognitor manual assay displayed analytical reactivity for a panel of the 20 most prevalent causes of bloodstream infection, with a detection range of 28-9050 CFU/mL. Validation with 1457 clinical blood cultures showed a negative predictive value of 99.0% compared to blood culture incubation for 5 days. Magnitor showed an improved detection range of 1-67 CFU/mL, allowing for detection of bacteria-supplemented blood cultures after 2-8 h incubation, and Candida albicans-supplemented blood cultures at 16-22 h, 5-15 h faster than blood culture. Removing an aliquot from a blood culture bottle and replacing the bottle into the incubator was shown not to result in contaminating organisms being introduced. CONCLUSIONS: The described method displays excellent breadth and detection for microbial cells and demonstrates the capability of confirming negative blood cultures after an overnight incubation in a blood culture instrument.

A quantitative analysis of kinase inhibitor selectivity.

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.

Optimal sensorimotor transformations for balance.

Here we have identified a sensorimotor transformation that is used by a mammalian nervous system to produce a multijoint motor behavior. Using a simple biomechanical model, a delayed-feedback rule based on an optimal tradeoff between postural error and neural effort explained patterns of muscle activation in response to a sudden loss of balance in cats. Following the loss of large sensory afferents, changes in these muscle-activation patterns reflected an optimal reweighting of sensory feedback gains to minimize postural instability. Specifically, a loss of center-of-mass-acceleration information, which allowed for a rapid initial rise in the muscle activity in intact animals, was absent after large-fiber sensory neuropathy. Our results demonstrate that a simple and flexible neural feedback control strategy coordinates multiple muscles over time via a small set of extrinsic, task-level variables during complex multijoint natural movements.

Adult mouse brain gene expression patterns bear an embryologic imprint.

The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional "imprint" consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior-posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org).

Sensitivity of the psychological and interpersonal relationship scales to oral therapies for erectile dysfunction.

INTRODUCTION: Studies of erectile dysfunction (ED) therapies typically assess erectile function end- points, although other treatment outcomes may also be of value to men and their partners. Aim. To examine the treatment sensitivity of the Psychological and Interpersonal Relationship Scales (PAIRS) associated with the use of two phosphodiesterase type 5 (PDE5) inhibitors with different duration of action. METHODS: Men with ED were recruited from three clinical trials of tadalafil and sildenafil citrate to complete post-treatment questionnaires that included the PAIRS. In study 1, the PAIRS was assessed after 6 months of open-label tadalafil (20 mg) treatment and after 2 months of real-world sildenafil use (25-100 mg). In studies 2 and 3, the PAIRS was assessed at the end of the trial assessment phase. In study 2, men with a 6-24 week history of sildenafil use (25-100 mg) received 3 weeks of open-label sildenafil treatment (dosage at study entry), and then switched to 9 weeks of tadalafil treatment. In study 3, a double-blind, crossover study, men were randomized to 12 weeks of either tadalafil (20 mg) or sildenafil (50 mg) treatment, followed by 12 weeks of the alternate treatment; PAIRS scores for the second treatment period were evaluated. MAIN OUTCOME MEASURE: Treatment outcomes were assessed with the PAIRS, a new reliable and valid self-report measure of sexual self-confidence, spontaneity, and time concerns before sex. RESULTS: Significant mean differences were observed on all PAIRS domain scores associated with tadalafil and sildenafil treatment. Across studies, men had significantly higher sexual self-confidence and spontaneity scores, and lower time concerns scores in reference to tadalafil compared with their scores in reference to sildenafil. CONCLUSIONS: Mean differences observed on PAIRS domains across studies support the sensitivity of the measure to differentiate between these two ED treatments.

Drivers and barriers to seeking treatment for erectile dysfunction: a comparison of six countries.

OBJECTIVE: To identify predictors of treatment-seeking behaviour in men with erectile dysfunction (ED) and the predictors (correlates) of individual drivers and barriers to seeking treatment. Although the prevalence and epidemiology of ED have been reviewed, there is little information about the treatment-seeking behaviour of men with this disorder. SUBJECTS AND METHODS: Data from the Cross-National Survey on Male Health Issues conducted between March and September 2000 were assessed by multivariate analysis. A cohort of 32 644 men aged 20-75 years was recruited during visits to their physicians. The men completed a short screening questionnaire, covering their overall health, and prostate, urinary and erectile problems. Men identified as having ED completed a detailed follow-up questionnaire. Logistic regression methods were used to identify predictors of treatment-seeking behaviour, and individual drivers and barriers to seeking treatment. RESULTS: Most men with ED had not sought treatment. The analyses suggested that ED, in conjunction with a desire to have sex, was necessary for men to seek treatment. Men seeking treatment commonly identified themselves as self-motivated or that they were influenced by a spouse or sex partner. The youngest group (20-39 years) was least likely to seek treatment. Among those who did not seek treatment, younger men were likely to believe that their ED would resolve spontaneously, whereas older men resisted seeking treatment because they felt that ED was a natural part of ageing. CONCLUSIONS: The data from this survey of men using the healthcare system confirmed other population-based reports that a minority of men with ED seek treatment. Subset analyses showed that treatment-seeking behaviour tended to be driven primarily by the man or by his sex partner. Common barriers to seeking treatment included the belief that ED would resolve spontaneously (younger men) and that ED was a normal part of ageing (older men).

A multicenter, randomized, double-blind, crossover study to evaluate patient preference between tadalafil and sildenafil.

PURPOSE: To assess patient preference for erectile dysfunction treatment between either sildenafil or tadalafil, each administered with their respective dosing instructions, and to evaluate preference for either sildenafil or tadalafil dosing instructions during tadalafil therapy. METHODS: We conducted a randomized, double-blind, crossover study consisting of four treatment arms. Because the dosing instructions for sildenafil and tadalafil are different, a unique methodology using sham placebo arms was employed to maintain the blind. To assess drug preference, 219 patients were randomized to either sildenafil 50 mg or tadalafil 20 mg, with dosing instructions reflecting their respective product profiles. To assess dosing instruction preference during tadalafil therapy, 46 patients were randomized to tadalafil 20 mg with either tadalafil or sildenafil dosing instructions. After 12 weeks, patients were crossed-over. After 4 weeks of each treatment, all patients following sildenafil dosing instructions were offered the opportunity for an upward dose titration. In a double-blind fashion, all patients who requested an upward titration received additional capsules. To mimic the pattern of dose usage observed in clinical practice, the number of patients who received additional double-blind active medication was limited to 35% of patients taking sildenafil in each treatment period in each country. Following the crossover treatment period, patients chose their preferred double-blind treatment with dosing instructions to receive in the 12-week extension period. RESULTS: In the drug preference assessment, 132 of 181 (73%) evaluable patients chose to receive tadalafil (p < 0.001) during the extension period. In the dosing instruction preference assessment, 24 of 36 (67%) evaluable patients preferred tadalafil with tadalafil dosing instructions (p = 0.046). Sildenafil and tadalafil were well tolerated. CONCLUSIONS: In the doses utilized in this study, 73% of patients preferred tadalafil with tadalafil dosing instructions for the treatment of their erectile dysfunction over sildenafil with sildenafil dosing instructions. During tadalafil therapy, 67% of patients preferred tadalafil dosing instructions over sildenafil dosing instructions.

The psychological and interpersonal relationship scales: assessing psychological and relationship outcomes associated with erectile dysfunction and its treatment.

Erectile dysfunction (ED) is associated with complex psychological and interpersonal issues. A new measure of treatment outcome, the Psychological and Interpersonal Relationship Scales (PAIRS), was developed to evaluate the broader psychological and interpersonal outcomes associated with erectile dysfunction and its treatment. Initial items were generated based on literature review, focus groups and market research, interviews with patients and partners, and consultation with expert clinicians. Domains of Sexual Self-Confidence, Spontaneity, and Time Concerns were identified and subsequently confirmed by factor analysis. A series of validation studies was performed with four separate samples, including assessment of internal consistency and test-retest reliability, convergent and discriminant validity, and treatment responsiveness. Samples for these studies included men recruited from clinical trials for ED in several countries ( N =413) and a community sample ( N =801). Findings from these studies indicate that PAIRS demonstrates adequate psychometric properties and appears well suited for use in further clinical studies of treatments for ED. It provides a broader assessment of treatment outcome than current measures of erectile function.

Reactions to merit pay increases: a longitudinal test of a signal sensitivity perspective.

The relationships among merit pay raises, trait positive affectivity (PA), and reactions to merit pay increases (pay attitudes and behavioral intentions) were explored in a longitudinal study of hospital employees. Drawing on signal sensitivity theory, the authors expected that PA would moderate the relationship between merit pay raise size and reactions to the increase such that pay raise size would be more strongly related to pay attitudes and behavioral intentions among those low in PA. Results strongly supported the predictions in the case of reactions to the raise amount (happiness and effort intentions) but not for pay level satisfaction. Implications of the results and directions for future research are identified.

Software and methods for oligonucleotide and cDNA array data analysis.

Two HTML-based programs were developed to analyze and filter gene-expression data: 'Bullfrog' for Affymetrix oligonucleotide arrays and 'Spot' for custom cDNA arrays. The programs provide intuitive data-filtering tools through an easy-to-use interface. A background subtraction and normalization program for cDNA arrays was also built that provides an informative summary report with data-quality assessments. These programs are freeware to aid in the analysis of gene-expression results and facilitate the search for genes responsible for interesting biological processes and phenotypes.