Julien Dupouey

Molecular detection of human rhinoviruses in respiratory samples: a comparison of Taqman probe-, SYBR green I- and BOXTO-based real-time PCR assays

BackgroundHuman Rhinoviruses (HRV) are major causative agents of acute respiratory tract infections in all age group and important contributing factors of childhood morbidity and mortality. Clinical presentation is poorly specific and the great antigenic and genetic variability of HRVs renders the biological diagnosis complex. Here, we have evaluated several molecular diagnostic protocols, including Taqman probe-based and intercalating agent-based RT-PCR assays.Methods5,627 respiratory samples sent to the laboratory of Virology of the University Hospitals of Marseille, France, from March 2011 to February 2012, were tested using a real-time RT-PCR assay in the 5’NCR of the rhinoviral genome that associated a Taqman probe and the detection of DNA-BOXTO-dye complexes. A sample of 500 BOXTO-positive samples were further tested using the same probe assay (without BOXTO), and a SYBR Green assay (using the same amplification primers). The specific amplification of HRV sequences was assessed by NGS amplicon sequencing.ResultsThe Taqman probe RT-PCR assay identified 696/5,627 samples (12,4%) as HRV-positive. BOXTO-positive samples included all probe-positive samples and 1,913 additional samples, of which only 24.3% were confirmed by sequencing. The SYBR Green assay was more specific (16/550 samples were probe-negative/SYBR Green-positive, all confirmed by 5′NCR sequencing), but 3/500 samples were probe-positive/SYBR Green-negative.ConclusionsOur results highlight the difficulty in detecting HRVs in clinical samples using a single molecular detection system. Amongst the 3 systems tested, the best compromise was obtained with the SYBR Green assay, which, by comparison with our probe-based assay provided an improved sensitivity without altering the detection specificity. Interestingly, a majority of probe-negative/BOXTO- or SYBR Green-positive samples were not associated with mutations in the sequence targeted by the probe. Sequence-based modifications of the secondary structure of the HRV 5′NCR may be associated with a limited access to the probe hybridisation region. Further investigations may identify a test combining a probe based- and an intercalating agent-based detection, which will significantly improve the diagnosis of HRV infections.

Pediatric Patients With Solid or Hematological Tumor Disease: Vancomycin Population Pharmacokinetics and Dosage Optimization.

Background: In pediatric cancer patients, determination of optimal vancomycin dosage is essential because of high risk of inadequate concentrations and bacterial resistance. The aim of this study was to determine vancomycin pharmacokinetic parameters in this population and propose dosage optimization to achieve optimal concentration. Methods: We retrospectively reviewed the use of vancomycin in pediatric cancer patients with febrile neutropenia (hematological or solid tumor disease). Vancomycin was administered by continuous infusion, and dosages were adapted according to therapeutic drug monitoring results. Blood cultures were performed before the first dose of antibiotic. Vancomycin pharmacokinetic population parameters were determined using NONMEM software, and dosage simulations were performed according to the target concentration (20–25 mg/L). Results: One hundred twenty-one patients were included in this study, representing 301 vancomycin concentrations. Blood cultures were positive in 37.5% of patients, and observed pathogens were mainly Staphylococcus spp. (43.8% methicillin resistant). Volume of distribution (95% confidence interval) was 34.7 L (17.3–48.0), and total apparent clearance (CL) (95% confidence interval) was correlated to body weight, tumor disease, and cyclosporine coadministration: CL = &thgr;CL × (WT/70)0.75 L/h with &thgr;CL = 3.49 (3.02–3.96), 4.66 (3.98–5.31), and 4.97 (4.42–5.41) in patients managed for hematological malignancies with or without cyclosporine coadministration and for solid malignancies, respectively. Based on simulation results, vancomycin dosage (milligram per kilogram) should be adapted to each child on the basis of its body weight and cyclosporine coadministration. Conclusions: Our results highlight the requirement to adapt vancomycin dosage in cancer pediatric population. Simulations have allowed to describe new dosage schedules, and a chart was created for clinicians to adapt vancomycin dosage.

Population pharmacokinetics of rifampicin in adult patients with osteoarticular infections: interaction with fusidic acid

Aims Rifampicin represents the key antibiotic for the management of osteoarticular infections. An important pharmacokinetic variability has already been described, particularly for absorption and metabolism. All previous pharmacokinetic studies have been focused only on patients treated for tuberculosis. The objective of the present study was to describe a population pharmacokinetic model of rifampicin in patients with staphylococcal osteoarticular infections, which has not been investigated to date. Method Rifampicin concentrations were collected retrospectively from 62 patients treated with oral rifampicin 300 mg three times daily. Plasma concentration–time data were analysed using NONMEM to estimate population pharmacokinetic parameters. Demographic data, infection characteristics and antibiotics taken in addition to rifampicin antibiotics were investigated as covariates. Results A one‐compartment model, coupled to a transit absorption model, best described the rifampicin data. Fusidic acid coadministration was identified as a covariate in rifampicin pharmacokinetic parameters. The apparent clearance and apparent central volume of distribution mean values [95% confidence interval (CI)] were 5.1 1 h–1 (1.2, 8.2 1 h–1)/23.8 l (8.9, 38.7 l) and 13.7 1 h–1 (10.6, 18.0 1 h–1)/61.1 1 (40.8, 129.0 1) for patients with and without administration of fusidic acid, respectively. Interindividual variability (95% CI) in the apparent clearance and apparent central volume of distribution were 72.9% (49.5, 86.0%) and 59.1% (5.5, 105.4%), respectively. Residual variability was 2.3 mg l–1 (1.6, 2.6 mg l–1). Conclusion We developed the first population pharmacokinetic model of rifampicin in patients with osteoarticular infections. Our model demonstrated that fusidic acid affects rifampicin pharmacokinetics, leading to potential high drug exposure. This finding suggests that fusidic acid dosing regimens should be reconsidered.

Simultaneous determination of four antiepileptic drugs in human plasma samples using an ultra-high-performance liquid chromatography tandem mass spectrometry method and its application in therapeutic drug monitoring

Therapeutic drug monitoring of antiepileptic drugs is widely practiced to achieve optimal efficacy and avoid adverse side effects. We describe an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS/MS) method developed for the monitoring of four frequently prescribed antiepileptic drugs - lamotrigine, levetiracetam, oxcarbazepine and topiramate. The main pharmacologically active metabolite of oxcarbazepine (mono-hydroxy-derivative metabolite, MHD) was also quantified. After addition of internal standards and a simple stage of protein precipitation, plasmatic samples were analyzed on a C18 column. All antiepileptic drugs were separated and quantified in 6 min, without interference. A good linearity was observed all over the calibration range (r2  > 0.99), up to 20 μg/mL (40 μg/mL for MHD). The limit of quantification was 0.20 μg/mL (0.40 μg/mL for MHD) with precision and accuracy ranging from 1.0 to 2.1% and from 96.7 to 110.8%, respectively. Intra- and inter-day precision and accuracy values were within 15%. No significant matrix effect was observed for all analytes. Clinical application was successfully evaluated in 259 samples from patients treated for epilepsy or bipolar disorders. In conclusion, a rapid, specific and sensitive UHPLC/MS/MS method was developed and validated for simultaneous quantification of antiepileptic drugs, suitable for therapeutic drug monitoring in neurology and psychiatry.

Pharmacogenetics of antidepressant drugs: State of the art and clinical implementation – recommendations from the French National Network of Pharmacogenetics

Tailoring antidepressant drug therapy to each individual patient is a complex process because these drugs have adverse effects leading to discontinuation. Pharmacogenetics may provide useful information in routine practice for optimizing antidepressant treatment by helping limit toxic effects while maintaining efficacy. This review presents the usefulness of pharmacogenetic tests for P450 cytochromes CYP2C19 and CYP2D6 in psychiatric patients taking antidepressants. Depending on the level of evidence, the French National Network of Pharmacogenetics (RNPGx) has issued recommendations stating that pharmacogenetic tests for CYP2D6 and CYP2C19 genes are potentially useful in psychiatric patients treated with antidepressant drugs.

Cannabis smoking impairs driving performance on the simulator and real driving: a randomized, double-blind, placebo-controlled, crossover trial

Driving experiments in real conditions are considered as a ‘gold standard’ to evaluate the effects of drugs on driving performance. Several constraints are difficult to manage in these conditions, so driving simulation appears as the best alternative. A preliminary comparison is crucial before being able to use driving simulation as a valid evaluation method. The aim of this study was to design a driving simulation method for assessing drug effects on driving. We used cannabis (THC) as a positive control and assessed whether THC affects driving performance in simulation conditions and whether these effects are consistent with performance in real driving conditions. A double‐blind, placebo‐controlled, two successive two‐way crossover design was performed using cigarettes containing 20 mg of THC. Healthy occasional users of THC, aged 25–35 years, who had a consistent driving experience were included. The first two sessions were realized in simulation conditions, and the last two sessions were in real driving conditions. Driving performance was estimated through inappropriate line crossings (ILC) and the standard deviation of the vehicles lateral position. Participants felt significantly drowsier and more tired after THC, whatever the driving condition. Driving stability was significantly impaired after THC, both in simulated and real driving conditions. We also found that ILC were significantly more numerous in driving simulation conditions, as compared to real driving. In conclusion, the driving simulator was proven to be more sensitive for demonstrating THC‐induced effects on driving performances. Driving simulation appears to be a good qualitative predictor of driving safety after drug intake.

Analytical interference during cefepime therapeutic drug monitoring in intensive care patient: About a case report

β-lactams therapeutic drug monitoring (TDM) appears as an essential tool to ensure the achievement of pharmacokinetic-pharmacodynamic targets and prevent induced toxicity in intensive care unit patients. Indeed, those patients exhibit important pharmacokinetic variabilities that could lead to unpredictable plasma concentrations, potentially associated with poor clinical outcome, development of antibiotic resistance or increased side effects. Here, we report the case of a 48-year-old-patient admitted to intensive care unit and treated by cefepime using TDM. Due to inconsistency between observed cefepime plasma concentrations and patient clinical examination, investigations were started. After analytical tests, we highlighted an underlying analytical interference that overestimated cefepime plasma concentration with our in-house high performance liquid chromatography with ultraviolet detection (HPLC-UV) method. Only the inadequacy between plasmatic concentration and patient situation alerted pharmacologists and clinicians. As we found no previous case in literature, we believe this report must serve as an example of analytical limits that required pharmacologist awareness and expertise in TDM realization.

ADFLAG ® , A DIAGNOSTIC BLOOD TEST FOR PRE-DEMENTIA STAGES OF ALZHEIMER’S DISEASE

P3-214 ADFLAG , A DIAGNOSTIC BLOOD TEST FOR PRE-DEMENTIA STAGES OF ALZHEIMER’S DISEASE Beatrice Blanc, Nicolas Pelletier, Clotilde Biscarrat, Pauline Picamal, Nathalie Compagnone, Samantha Galluzzi, Moira Marizzoni, Jorge Jovicich, Giovanni B. Frisoni, Gianluigi Forloni, Diego Albani, Jill Richardson, Lucilla Parnetti, Magda Tsolaki, Flavio Nobili, David Bartrez-faz, Mira Didic, Peter Schoenknecht, Pierre Payoux, Andrea Soricelli, Paolo Rossini, Pieter Jelle Visser, Regis Bordet, Ute Fiedler, Olivier Blin, Julien Dupouey, Joelle Micallef, Laura Lanteaume, Bernard Michel, ICDD, Gemenos, France; IRCCS Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; University of Trento, Trento, Italy; Memory Clinic and LANVIE Laboratory of Neuroimaging of Aging, University Hospitals and University of Geneva, Geneva, Switzerland; Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy; IRCCS, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; GSK R&D, China-United Kingdom, Middlesex, United Kingdom; Lab of Clinical Neurochemistry, University of Perugia, Perugia, Italy; Aristotle University of Thessaloniki, Thessaloniki, Greece; University of Genoa, Italy, Genoa, Italy; Department of Psychiatry and Clinical Psychobiology, Faculty of Medicine, University of Barcelona and Institut d’Investigacions Biom ediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Service de Neurologie et Neuropsychologie, Marseille, France; Department of Psychiatry and Psychotherapy, University of Leipzig, Leipzig, Germany; INSERM, Imagerie C er ebrale et Handicaps Neurologiques, Toulouse, France; Fondazione SDN per la Ricerca e l’Alta Formazione in Diagnostica Nucleare, Naples, Italy; Department of Gerontology, Neurosciences & Orthopedics, Catholic University, Rome, Italy; VU University Medical Center, Amsterdam, Netherlands; Service de Pharmacologie-Hôpital Huriez-CHRU, Lille, France; Institutes and Clinics of the University Duisburg-Essen, Essen, Germany; Mediterranean Institute of Cognitive Neurosciences, Marseille, France; Service de Neurologie et Neuropsychologie, CHU la Timone, Marseille, France; Assistance Publique Hôpitaux de Marseille, Marseille, France; Service de Neurologie Comportementale, Hôpital Sainte Marguerite, Marseille, France, CNRS LNIAUMR 7260 FR3C FR 3512, Aix-Marseille Universit e, Marseille, France. Contact e-mail: bblanc@icdd-sas.com

Translational research on cognitive and behavioural disorders in neurological and psychiatric diseases.

The important medical and social burden of nervous system diseases contrasts with the currently limited therapeutic armamentarium and with the difficulty encountered in developing new therapeutic options. These failures can be explained by the conjunction of various phenomena related to the limitations of animal models, the narrow focus of research on precise pathophysiological mechanisms, and methodological issues in clinical trials. It is perhaps the paradigm itself of the way research is conducted that may be the real reason for our incapacity to find effective strategies. The purpose of this workshop was to define overall lines of research that could lead to the development of effective novel therapeutic solutions. Research has long focused on diseases per se rather than on cognitive and behavioural dimensions common to several diseases. Their expression is often partial and variable, but can today be well-characterised using neurophysiological or imaging methods. This dimensional or syndromic vision should enable a new insight to the question, taking a transnosographic approach to re-position research and to propose: translational models exploring the same functions in animal models and in humans; identification of homogeneous groups of patients defined according to the clinical, anatomico-functional and molecular characteristics; and preclinical and clinical developments enriched by the use of cognitive-behavioural, biological neurological, and imaging biomarkers. For this mutation to be successful, it must be accompanied by synchronised action from the public authorities and by ad hoc measures from the regulatory agencies.

Concentrations systémiques d’antibiotiques potentiellement toxiques après administration par pansement imprégné chez un patient brûlé : à propos d’un cas clinique☆

Severe burned patients present high risk of skins infections, frequently due to Pseudomonas aeruginosa. Impregnated dressings with amikacin or colistin could be a good alternative to obtain effective concentration directly at the infected site. Therapeutic drug monitoring for these antibiotics is currently recommended after an intravenous administration to obtain effective and non-toxic plasmatic concentrations. However, data are lacking about systemic exposition and risk of toxicity after an administration with impregnated dressings. We report the case of a severe burned patient with cutaneous infection treated with amikacin and colistin impregnated dressings, for which plasmatic pharmacokinetic profiles were performed.