Population pharmacokinetics and toxicodynamics of continuously infused linezolid in critically ill patients.

OBJECTIVES: Linezolid is a treatment option against multi-drug-resistant Gram-positive pathogens. Continuous infusion of linezolid has been proposed to optimize antimicrobial exposure, although pharmacokinetic data from large patient cohorts are lacking. METHODS: Population pharmacokinetics and the time-dependent association between linezolid exposure and the occurrence of thrombocytopenia in 120 critically ill patients were described. Monte Carlo simulations evaluated pharmacokinetic/pharmacodynamic/toxicodynamic target attainment in relation to body weight and creatinine clearance for continuously infused doses of 300-2400 mg/day. RESULTS: Linezolid pharmacokinetics were highly variable (interindividual variability of clearance: 52.8% coefficient of variation). Non-linear clearance was quantified, which decreased from 6.82 to 3.82 L/h within 3-6 days in the population. A relationship between linezolid exposure and platelet count over time was established. For standard dosing (1200 mg/day), the model predicted Grade 2, 3 or 4 thrombocytopenia (<75 × 103/µL, <50 × 103/µL and <25 × 103/µL) in 21.7%, 10.4% and 2.5% of patients at day 14, respectively. Patients with impaired renal function displayed higher risk. The overall probability of Grade 3 thrombocytopenia could be reduced from 10.4% using standard dosing to 6.3% if a linezolid steady state plasma concentration of 7 mg/L is targeted, suggesting a value of therapeutic drug monitoring (TDM). CONCLUSION: Dosing linezolid by continuous infusion should include considerations of creatinine clearance and body weight to maximize the achievement of therapeutic exposures. However, due to the high variability in individual dose, optimization using TDM seems necessary to optimize linezolid dosing under continuous infusion to avoid toxicity, particularly if longer treatment courses are expected.

Antibiotic Stewardship and Therapeutic Drug Monitoring of ß-Lactam Antibiotics: Is There a Link? An Opinion Paper.

PURPOSE: In critically ill patients, changes in the pharmacokinetics (PK) of ß-lactams can lead to significant variations in serum concentrations, with possibly detrimental effects on outcomes. The utilization of individually calculated doses, extended infusion regimen, and therapeutic drug monitoring (TDM)-guided dose adjustments can mitigate the PK changes and help to achieve and attain an individual PK target. METHODS: We reviewed relevant literature from 2004 to 2021 using 4 search engines (PubMed, Web of Science, Scopus, and Google Scholar). Unpublished clinical data were also examined. RESULTS: TDM-guided, individualized dosing strategies facilitated PK target attainment and improved patient outcomes. TDM-guided therapy is a core concept of individualized dosing that increases PK target attainment and identifies possible toxic ß-lactam concentrations. CONCLUSIONS: Individualized dosing and TDM facilitate the rational use of ß-lactams and are integral for antibiotic stewardship interventions in critical care, affording the optimal exposure of both pathogen and drugs, along with enhanced treatment efficacy and reduced emergence of antimicrobial resistance.

Personalized Piperacillin Dosing for the Critically Ill: A Retrospective Analysis of Clinical Experience with Dosing Software and Therapeutic Drug Monitoring to Optimize Antimicrobial Dosing.

Optimization of antibiotic dosing is a treatment intervention that is likely to improve outcomes in severe infections. The aim of this retrospective study was to describe the therapeutic exposure of steady state piperacillin concentrations (cPIP) and clinical outcome in critically ill patients with sepsis or septic shock who received continuous infusion of piperacillin with dosing personalized through software-guided empiric dosing and therapeutic drug monitoring (TDM). Therapeutic drug exposure was defined as cPIP of 32-64 mg/L (2-4× the 'MIC breakpoint' of Pseudomonas aeruginosa). Of the 1544 patients screened, we included 179 patients (335 serum concentrations), of whom 89% achieved the minimum therapeutic exposure of >32 mg/L and 12% achieved potentially harmful cPIP > 96 mg/L within the first 48 h. Therapeutic exposure was achieved in 40% of the patients. Subsequent TDM-guided dose adjustments significantly enhanced therapeutic exposure to 65%, and significantly reduced cPIP > 96 mg/L to 5%. Mortality in patients with cPIP > 96 mg/L (13/21; 62%) (OR 5.257, 95% CI 1.867-14.802, p = 0.001) or 64-96 mg/L (30/76; 45%) (OR 2.696, 95% CI 1.301-5.586, p = 0.007) was significantly higher compared to patients with therapeutic exposure (17/72; 24%). Given the observed variability in critically ill patients, combining the application of dosing software and consecutive TDM increases therapeutic drug exposure of piperacillin in patients with sepsis and septic shock.

Therapeutic drug monitoring-guided continuous infusion of piperacillin/tazobactam significantly improves pharmacokinetic target attainment in critically ill patients: a retrospective analysis of four years of clinical experience.

PURPOSE: Standard dosing and intermittent bolus application (IB) are important risk factors for pharmacokinetic (PK) target non-attainment during empirical treatment with ß-lactams in critically ill patients, particularly in those with sepsis and septic shock. We assessed the effect of therapeutic drug monitoring-guided (TDM), continuous infusion (CI) and individual dosing of piperacillin/tazobactam (PIP) on PK-target attainment in critically ill patients. METHODS: This is a retrospective, single-center analysis of a database including 484 patients [933 serum concentrations (SC)] with severe infections, sepsis and septic shock who received TDM-guided CI of PIP in the intensive care unit (ICU) of an academic teaching hospital. The PK-target was defined as a PIP SC between 33 and 64 mg/L [fT > 2-4 times the epidemiological cutoff value (ECOFF) of Pseudomonas aeruginosa (PSA)]. RESULTS: PK-target attainment with standard dosing (initial dose) was observed in 166 patients (34.3%), whereas only 49 patients (10.1%) demonstrated target non-attainment. The minimum PK-target of ≥ 33 mg/L was overall realized in 89.9% (n = 435/484) of patients after the first PIP dose including 146 patients (30.2%) with potentially harmful SCs ≥ 100 mg/L. Subsequent TDM-guided dose adjustments significantly enhanced PK-target attainment to 280 patients (62.4%) and significantly reduced the fraction of potentially overdosed (≥ 100 mg/L) patients to 4.5% (n = 20/449). Renal replacement therapy (RRT) resulted in a relevant reduction of PIP clearance (CLPIP): no RRT CLPIP 6.8/6.3 L/h (median/IQR) [SCs n = 752, patients n = 405], continuous veno-venous hemodialysis (CVVHD) CLPIP 4.3/2.6 L/h [SCs n = 160, n = 71 patients], intermittent hemodialysis (iHD) CLPIP 2.6/2.3 L/h [SCs n = 21, n = 8 patients]). A body mass index (BMI) of > 40 kg/m2 significantly increased CLPIP 9.6/7.7 L/h [SC n = 43, n = 18 patients] in these patients. Age was significantly associated with supratherapeutic PIP concentrations (p < 0.0005), whereas high CrCL led to non-target attainment (p < 0.0005). Patients with target attainment (33-64 mg/L) within the first 24 h exhibited the lowest hospital mortality rates (13.9% [n = 23/166], p < 0.005). Those with target non-attainment demonstrated higher mortality rates (≤ 32 mg/L; 20.8% [n = 10/49] ≥ 64 mg/L; 29.4% [n = 79/269]). CONCLUSION: TDM-guided CI of PIP is safe in critically ill patients and improves PK-target attainment. Exposure to defined PK-targets impacts patient mortality while lower and higher than intended SCs may influence the outcome of critically ill patients. Renal function and renal replacement therapy are main determinants of PK-target attainment. These results are only valid for CI of PIP and not for prolonged or intermittent bolus administration of PIP.

[Diagnostic Approaches in Sepsis - Part 1: General Diagnostic Principles, Focus Identification and Source Control]. / Diagnostik der Sepsis ­ Teil 1: allgemeine Diagnostik und Fokussuche-/sanierung.

Sepsis and septic shock represent medical emergencies with persistently high mortality rates. According to the lately revised Surviving Sepsis Campaign (SSC) guidelines, focus identification/pathogen detection and the initial administration of broad-spectrum antibiotics are to be secluded within one hour after recognition of the symptoms of sepsis. However, there is dispute concerning the so called hour-1 bundle. Being a core aspect of focus identification, imaging modalities mainly depend on the suspected site of infection and the individual patient. Contrast agent-enhanced computed tomography (CT) is the modality usually used in critically ill patients. The microbiological pathogen detection still largely remains culture-based. This emphasizes the significance of microbiological specimen obtained from easily accessible body compartments and at least 2 blood culture sets. If possible, blood cultures should be drawn prior to antibiotic administration. Intraoperatively obtained swabs of otherwise sterile body compartments are of utmost importance with regard to microbiological pathogen detection. Catheters and implanted medical devices (i.e. cardiac pacemakers or defibrillators) suspicious of infection should be explanted and sent in for microbiological workup as soon as possible. All necessary source control measures should be realized as soon as medically possible but at least within 6 - (12) hours after the onset of symptoms. There is no specific biomarker for sepsis so far. Procalcitonin (PCT) and C-reactive protein (CRP) are crucial biomarkers in terms of infectious disease management and guidance of antimicrobial therapy in the ICU. Positive clinical trials showed that biomarkers like the midregional pro-adrenomedullin (MR-proADM) or presepsin might be promising candidates in the diagnosis of sepsis in the future. As an important marker of microcirculatory failure and disrupted cell metabolism, lactate serum concentrations (and lactate-clearance, respectively) are of prognostic value in septic patients.

[Diagnostic Approaches in Sepsis - Part 2: Pathogen Detection]. / Diagnostik der Sepsis ­ Teil 2: Erregeridentifikation.

Despite the dissemination of innovative, molecular biology-based and commercially available devices for pathogen detection, culture-based methods with susceptibility testing remain the key principles for guiding antimicrobial treatment of patients suffering from sepsis or septic shock on the ICU. Culture-based methods are able to facilitate pathogen detection from a diversity of specimen (respiratory secretion, intraoperatively obtained smears, aspirates, and so forth). However, the latency from obtainment of the specimen up to pathogen detection with susceptibility testing is a major disadvantage of culture-based methods in critical illness. Molecular biology-based methods like Polymerase Chain Reaction (PCR) and especially Next-Generation Sequencing (NGS) based methods promise faster pathogen and resistance detection, but are not used in clinical routine yet. With more clinical trials to come, these innovative diagnostic tools may have the potential to lead to a paradigm shift within the context of pathogen identification in sepsis.

Finding the needle in the haystack: differentiating "identical" twins in paternity testing and forensics by ultra-deep next generation sequencing.

Monozygotic (MZ) twins are considered being genetically identical, therefore they cannot be differentiated using standard forensic DNA testing. Here we describe how identification of extremely rare mutations by ultra-deep next generation sequencing can solve such cases. We sequenced DNA from sperm samples of two twins and from a blood sample of the child of one twin. Bioinformatics analysis revealed five single nucleotide polymorphisms (SNPs) present in the twin father and the child, but not in the twin uncle. The SNPs were confirmed by classical Sanger sequencing. Our results give experimental evidence for the hypothesis that rare mutations will occur early after the human blastocyst has split into two, the origin of twins, and that such mutations will be carried on into somatic tissue and the germline. The method provides a solution to solve paternity and forensic cases involving monozygotic twins as alleged fathers or originators of DNA traces.

Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays.

Herein, we describe how cantilever-free scanning probes can be used to deposit precursor material and subsequently irradiate the precursor to initiate polymerization, resulting in a 3D lithographic method wherein the position, height and diameter of each feature can be tuned independently. Specifically, acrylate and methacrylate monomers were patterned onto thiol terminated glass and subsequently exposed to UV light produced brush polymers by a photoinduced radical acrylate polymerization reaction. Here, we report the first examples of glycan arrays, comprised of methacrylate brush polymers that are side-chain functionalized with α-glucose, by this new lithographic approach. Their binding with fluorophore labeled concanavalin A (ConA) was assayed by fluorescence microscopy. The fluorescence of these brush polymers was compared to glycan arrays composed of monolayers of α-mannosides and α-glucosides prepared by combining polymer pen lithography (PPL) with the thiol-ene photochemical reaction or the copper-catalyzed azide-alkyne cycloaddition. At high ConA concentration, the fluorescence signal of the brush polymer was nearly 20 times greater than that of the glycan monolayers, and the brush polymer arrays had a detection limit nearly two orders of magnitude better than their monolayer counterparts. Because of the ability of this method to control precisely the polymer length, the relationship between limit of detection and multivalency could be explored, and it was found that the longer polymers (136 nm) are an order of magnitude more sensitive towards ConA binding than the shorter polymers (8 nm) and that binding affinity decreased systematically with length. These glycan arrays are a new tool to study the role of multivalency on carbohydrate recognition, and the photopolymerization route towards forming multivalent glycan scaffolds described herein, is a promising route to create multiplexed glycan arrays with nanoscale feature dimensions.

Comparative genomic analyses of the Taylorellae.

Contagious equine metritis (CEM) is an important venereal disease of horses that is of concern to the thoroughbred industry. Taylorella equigenitalis is a causative agent of CEM but very little is known about it or its close relative Taylorella asinigenitalis. To reveal novel information about Taylorella biology, comparative genomic analyses were undertaken. Whole genome sequencing was performed for the T. equigenitalis type strain, NCTC11184. Draft genome sequences were produced for a second T. equigenitalis strain and for a strain of T. asinigenitalis. These genome sequences were analysed and compared to each other and the recently released genome sequence of T. equigenitalis MCE9. These analyses revealed that T. equigenitalis strains appear to be very similar to each other with relatively little strain-specific DNA content. A number of genes were identified that encode putative toxins and adhesins that are possibly involved in infection. Analysis of T. asinigenitalis revealed that it has a very similar gene repertoire to that of T. equigenitalis but shares surprisingly little DNA sequence identity with it. The generation of genome sequence information greatly increases knowledge of these poorly characterised bacteria and greatly facilitates study of them.

Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG.

BACKGROUND: Metagenomics is the study of microbial organisms using sequencing applied directly to environmental samples. Technological advances in next-generation sequencing methods are fueling a rapid increase in the number and scope of metagenome projects. While metagenomics provides information on the gene content, metatranscriptomics aims at understanding gene expression patterns in microbial communities. The initial computational analysis of a metagenome or metatranscriptome addresses three questions: (1) Who is out there? (2) What are they doing? and (3) How do different datasets compare? There is a need for new computational tools to answer these questions. In 2007, the program MEGAN (MEtaGenome ANalyzer) was released, as a standalone interactive tool for analyzing the taxonomic content of a single metagenome dataset. The program has subsequently been extended to support comparative analyses of multiple datasets. RESULTS: The focus of this paper is to report on new features of MEGAN that allow the functional analysis of multiple metagenomes (and metatranscriptomes) based on the SEED hierarchy and KEGG pathways. We have compared our results with the MG-RAST service for different datasets. CONCLUSIONS: The MEGAN program now allows the interactive analysis and comparison of the taxonomical and functional content of multiple datasets. As a stand-alone tool, MEGAN provides an alternative to web portals for scientists that have concerns about uploading their unpublished data to a website.

Methods for comparative metagenomics.

BACKGROUND: Metagenomics is a rapidly growing field of research that aims at studying uncultured organisms to understand the true diversity of microbes, their functions, cooperation and evolution, in environments such as soil, water, ancient remains of animals, or the digestive system of animals and humans. The recent development of ultra-high throughput sequencing technologies, which do not require cloning or PCR amplification, and can produce huge numbers of DNA reads at an affordable cost, has boosted the number and scope of metagenomic sequencing projects. Increasingly, there is a need for new ways of comparing multiple metagenomics datasets, and for fast and user-friendly implementations of such approaches. RESULTS: This paper introduces a number of new methods for interactively exploring, analyzing and comparing multiple metagenomic datasets, which will be made freely available in a new, comparative version 2.0 of the stand-alone metagenome analysis tool MEGAN. CONCLUSION: There is a great need for powerful and user-friendly tools for comparative analysis of metagenomic data and MEGAN 2.0 will help to fill this gap.

MetaSim: a sequencing simulator for genomics and metagenomics.

BACKGROUND: The new research field of metagenomics is providing exciting insights into various, previously unclassified ecological systems. Next-generation sequencing technologies are producing a rapid increase of environmental data in public databases. There is great need for specialized software solutions and statistical methods for dealing with complex metagenome data sets. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate the development and improvement of metagenomic tools and the planning of metagenomic projects, we introduce a sequencing simulator called MetaSim. Our software can be used to generate collections of synthetic reads that reflect the diverse taxonomical composition of typical metagenome data sets. Based on a database of given genomes, the program allows the user to design a metagenome by specifying the number of genomes present at different levels of the NCBI taxonomy, and then to collect reads from the metagenome using a simulation of a number of different sequencing technologies. A population sampler optionally produces evolved sequences based on source genomes and a given evolutionary tree. CONCLUSIONS/SIGNIFICANCE: MetaSim allows the user to simulate individual read datasets that can be used as standardized test scenarios for planning sequencing projects or for benchmarking metagenomic software.

Dendroscope: An interactive viewer for large phylogenetic trees.

BACKGROUND: Research in evolution requires software for visualizing and editing phylogenetic trees, for increasingly very large datasets, such as arise in expression analysis or metagenomics, for example. It would be desirable to have a program that provides these services in an efficient and user-friendly way, and that can be easily installed and run on all major operating systems. Although a large number of tree visualization tools are freely available, some as a part of more comprehensive analysis packages, all have drawbacks in one or more domains. They either lack some of the standard tree visualization techniques or basic graphics and editing features, or they are restricted to small trees containing only tens of thousands of taxa. Moreover, many programs are difficult to install or are not available for all common operating systems. RESULTS: We have developed a new program, Dendroscope, for the interactive visualization and navigation of phylogenetic trees. The program provides all standard tree visualizations and is optimized to run interactively on trees containing hundreds of thousands of taxa. The program provides tree editing and graphics export capabilities. To support the inspection of large trees, Dendroscope offers a magnification tool. The software is written in Java 1.4 and installers are provided for Linux/Unix, MacOS X and Windows XP. CONCLUSION: Dendroscope is a user-friendly program for visualizing and navigating phylogenetic trees, for both small and large datasets.

OSLay: optimal syntenic layout of unfinished assemblies.

UNLABELLED: The whole genome shotgun approach to genome sequencing results in a collection of contigs that must be ordered and oriented to facilitate efficient gap closure. We present a new tool OSLay that uses synteny between matching sequences in a target assembly and a reference assembly to layout the contigs (or scaffolds) in the target assembly. The underlying algorithm is based on maximum weight matching. The tool provides an interactive visualization of the computed layout and the result can be imported into the assembly editing tool Consed to support the design of primer pairs for gap closure. MOTIVATION: To enhance efficiency in the gap closure phase of a genome project it is crucial to know which contigs are adjacent in the target genome. Related genome sequences can be used to layout contigs in an assembly. AVAILABILITY: OSLay is freely available from: http://www-ab.informatik.unituebingen.de/software/oslay.