First description of blaNDM-1, blaOXA-48, blaOXA-181 producing Enterobacteriaceae strains in Romania

We report the first isolation and characterization of several Enterobacteriaceae strains harboring bla_NDM-1, bla_OXA-48 and/or bla_OXA-181 genes in a Romanian emergency teaching hospital. Between January 2010 and September 2012 nine carbapenemase-producing Enterobacteriaceae strains were identified. The bla_NDM-1 gene was present in two Enterobacter cloacae strains, an Escherichia coli and two Klebsiella pneumoniae strains. One of these K. pneumoniae strains also harbored the bla_OXA-181 gene. Three other K. pneumoniae strains and one Serratia marcescens carried bla_OXA-48.     

Fitness cost associated with resistance to fluoroquinolones is diverse across clones of Klebsiella pneumoniae and may select for CTX-M-15 type extended-spectrum β-lactamase

Lowered fitness cost associated with resistance to fluoroquinolones was recently demonstrated to influence the clonal dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in the health care setting. We investigated whether or not a similar mechanism impacts Klebsiella pneumoniae. The fitness of K. pneumoniae isolates from major international hospital clones (ST11, ST15, ST147) already showing high-level resistance to fluoroquinolones and of strains from three minor clones (ST25, ST274, ST1028) in which fluoroquinolone resistance was induced in vitro was tested in a propagation assay. Strains from major clones showed significantly less fitness cost than three of four fluoroquinolone-resistant derivatives of minor clone isolates. In addition, plasmids with CTX-M-15 type extended-spectrum β-lactamase (ESBL) genes were all retained in both major and minor clone isolates, irrespective of the strains’ level of fluoroquinolone resistance, while each plasmid harboring SHV-type ESBLs had been lost during the induction of resistance. Major clone K. pneumoniae strains harbored more amino acid substitutions in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes than minor clone isolates. The presence of an active efflux system could be demonstrated in all fluoroquinolone-resistant derivatives of originally SHV-producing minor clone isolates but not in any CTX-M-15-producing strain. Further investigations are needed to expand and confirm our findings on a larger sample. In addition, a long-term observation of our ciprofloxacin-resistant minor clone isolates is required in order to elucidate whether or not they are capable of restoring their fitness while concomitantly retaining high minimum inhibitory concentration (MIC) values.     

Development of a Whole Body Physiologically Based Model to Characterise the Pharmacokinetics of Benzodiazepines. 1: Estimation of Rat Tissue-Plasma Partition Ratios

Three methods for estimation of the equilibrium tissue-to-plasma partition ratios (Kp values) in the presence of tissue concentration time data have been investigated. These are the area method, the open loop (tissue specific) method and the whole body model(closed loop) method, each with different model assumptions. Additionally, multiple imputations, a technique for dealing with deficiencies in data sets (i.e., missing tissues) is used. The estimated Kp values by the three methods have been compared and the limitations and advantages of each approach drawn. The area method, which is essentially model free, gives only a crude estimate of Kp without making any statement of its uncertainty; whereas both the open and closed loop methods provide an estimate of this. The closed loop method, where the most assumptions are made, is the approach that gives the best overall estimates of Kp, which was confirmed by comparing the predicted concentration-time profiles with experimental data. Although the estimates from the closed loop method, as well as the other two methods, are conditioned on the data, they are the most reliable for both propagating parameter variability and uncertainty through a whole body physiologically based model, as well as for extrapolation to human. A series of benzodiazepines, namely alprazolam, chlordiazepoxide, clobazam, diazepam, flunitrazepam, midazolam and triazolam in rat is used as a case study in the current investigation.     

Incorporating measures of variability and uncertainty into the prediction of in vivo hepatic clearance from in vitro data.

The existing procedures for quantitative in vitro-in vivo clearance prediction can be significantly biased either by totally neglecting the existing variability and uncertainty by using mean parameter values or by implementing Monte Carlo simulation with statistical distribution of the parameters reconstructed from very small sets of data. The aim of the present study is to develop a methodology for the prediction of in vivo hepatic clearance in the presence of semiquantitative or qualitative data and accounting for the existing uncertainty and variability. The method consists of two steps: 1) transformation of the information available into fuzzy sets (fuzzification); and 2) computation of the in vivo clearance using arithmetic operations with fuzzy sets. To illustrate the approach, rat hepatocyte and microsomal data for eight benzodiazepine compounds are used. A comparison with a standard Monte Carlo procedure is made. The methodology proposed can be used when Monte Carlo simulation may be biased or cannot be implemented. The obtained fuzzy in vivo clearance can be used subsequently in fuzzy simulations of pharmacokinetic models.     

Reducing Whole Body Physiologically Based Pharmacokinetic Models Using Global Sensitivity Analysis: Diazepam Case Study

Abtract There are situations in drug development where one may wish to reduce the dimensionality and complexity of whole body physiologically based pharmacokinetic models. A technique for formal reduction of such models, based on global sensitivity analysis, is suggested. Using this approach mean and variance of tissue(s) and/or blood concentrations are preserved in the reduced models. Extended Fourier amplitude sensitivity test (FAST), a global sensitivity technique, takes a sampling approach, acknowledging parameter variability and uncertainty, to calculate the impact of parameters on concentration variance. We used existing literature rules for formal model reduction to identify all possible smaller dimensionally models. To discriminate among those competing mechanistic models extended FAST was used, whereby we treated model structural uncertainty as another factor contributing to the overall uncertainty. A previously developed 14 compartment whole body physiologically based model for diazepam disposition in rat was reduced to three alternative reduced models, with preserved arterial mean and variance concentration profiles.     

Diazepam Pharamacokinetics from Preclinical to Phase I Using a Bayesian Population Physiologically Based Pharmacokinetic Model with Informative Prior Distributions in Winbugs

Modelling is an important applied tool in drug discovery and development for the prediction and interpretation of drug pharmacokinetics. Preclinical information is used to decide whether a compound will be taken forwards and its pharmacokinetics investigated in human. After proceeding to human little to no use is made of these often very rich data. We suggest a method where the preclinical data are integrated into a whole body physiologically based pharmacokinetic (WBPBPK) model and this model is then used for estimating population PK parameters in human. This approach offers a continuous flow of information from preclinical to clinical studies without the need for different models or model reduction. Additionally, predictions are based upon single parameter values, but making realistic predictions involves incorporating the various sources of variability and uncertainty. Currently, WBPBPK modelling is undertaken as a two-stage process: (i) estimation (optimisation) of drug-dependent parameters by either least squares regression or maximum likelihood and (ii) accounting for the existing parameter variability and uncertainty by stochastic simulation. To address these issues a general Bayesian approach using WinBUGS for estimation of drug-dependent parameters in WBPBPK models is described. Initially applied to data in rat, this approach is further adopted for extrapolation to human, which allows retention of some parameters and updating others with the available human data. While the issues surrounding the incorporation of uncertainty and variability within prediction have been explored within WBPBPK modeling methodology they have equal application to other areas of pharmacokinetics, as well as to pharmacodynamics.     

Molecular characterization of Campylobacter lanienae strains isolated from food-producing animals

During 2008 and 2009, within the framework of the Hungarian monitoring program of antibiotic resistance of zoonotic agents from food-producing animals, a significant number (43 strains) of Campylobacter lanienae were detected for the first time in Hungary. The isolates were genotyped using partial 16S rRNA gene sequencing and pulsed-field gel electrophoresis using three different restriction enzymes. The antimicrobial resistance of the isolates was determined by microtiter broth dilution. C. lanienae isolation was successful only from swine but not from other animal species. According to phylogenetic analysis, clustering of the isolates shows the same extensive genetic diversity as other Campylobacter species. Sequence analysis of the partial 16S rRNA gene showed that additional variations exist in variable regions Vc2 and Vc6. SmaI restriction enzyme proved to be the most efficient for pulsed-field gel electrophoresis analysis of C. lanienae. A significant tetracycline resistance (60.9%) and the presence of erythromycin-, enrofloxacin-, and multiresistant C. lanienae strains were found. Although the pathogenic potential of C. lanienae in humans is currently unknown, this study demonstrates that C. lanieanae is common in pigs in the country, provides further details on the genotypic and phenotypic properties of C. lanienae, and offers a genotyping method for use in source tracing.     

Emergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clone in Hungary

Nine Klebsiella pneumoniae isolates showing non-susceptibility to carbapenems were collected from three centres in the north-eastern region of Hungary. The minimum inhibitory concentrations (MICs) of antibiotics were determined by Etest. The putative production of a carbapenemase was tested by the modified Hodge test. The presence of bla _KPC genes was verified by polymerase chain reaction (PCR) and sequencing. Furthermore, molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates showed extensively drug-resistant (XDR) phenotype, and of these, eight isolates were highly resistant to colistin. The isolates carried bla _KPC-2, bla _SHV-12, bla _TEM-1 and bla _SHV-11. PFGE analysis of the nine KPC-2-producing Hungarian ST258 K. pneumoniae isolates, two KPC-2-producing Norwegian ST258 isolates and 33 CTX-M-15-producing ST11 isolates revealed the existence of one genetic cluster at an 88% similarity level. The overall results of the PFGE clustering, MLST and the presence of SHV-11 in both ST11 and ST258 suggest that this is the first hyperepidemic clonal complex of multidrug-resistant K. pneumoniae, probably CC258/CC340, possibly undergoing worldwide spread.     

Evaluation of acute/late toxicity and local recurrence in T1–T2 glottic carcinoma treated with accelerated hypofractionated 3D-conformal external beam radiotherapy (3D-CRT)

Background

The aim of the study was to evaluate the efficacy, as well as the acute and late toxicity of an accelerated hypofractionated 3DCRT schedule as radical treatment in patients with organ confined glottic cancer cT1-2N0.

Patients and methods.

Between June of 2004 and September 2010, 47 retrospectively selected patients (29 males, 18 females) diagnosed with organ confined T1 or T2 glottic cancer, were treated with external 3DCRT in an accelerated hypofractionation schedule. The median age was 70 years. A dose of 64.4 Gy in 28 daily fractions was prescribed. The primary study endpoints were to assess the acute and late effects of radiation toxicity, according to the EORTC/ RTOG scale, as well as the therapeutic impact of this schedule in terms of local recurrence.

Results

The median follow up was 36 months. At the end of radiotherapy, grade I, II and III acute toxicity was observed in 34, 9 and4 patients, respectively. Late grade I and II toxicity was observed in 25 and in 8 patients respectively. Only two local recurrences were observed, 15 and 24 months post 3DCRT respectively.

Conclusions

Our radiotherapy schedule achieves a high locoregional control rate with the advantage of voice preservation. The proposed hypofractionated schedule can be recommended as a standard radiotherapy treatment, since these results are comparable with those of conventional fractionation schedules.