Extended multilocus variable-number tandem-repeat analysis of Clostridium difficile correlates exactly with ribotyping and enables identification of hospital transmission.

PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.

A realist evaluation of a collaborative model to support research co-production in long-term care settings in England: the ExCHANGE protocol.

BACKGROUND: Collaborative working between academic institutions and those who provide health and social care has been identified as integral in order to produce acceptable, relevant, and timely research, and for outputs to be useful and practical to implement. The ExCHANGE Collaboration aims to bring together researchers and people working, living in and visiting care homes to build capacity, share and mobilise knowledge, and identify key areas for future research. This paper describes an embedded, formative, realist and theory-driven evaluation which aims to gather information about how successful the ExCHANGE Collaboration is perceived to be in achieving its aims. An existing realist programme theory from the literature - Closer Collaboration - will be supplemented by two substantive theories: Co-production and Knowledge Brokering. This will result in an initial programme theory which will be tested by this formative evaluation to refine understanding of how the ExCHANGE Collaboration works. METHODS: The evaluation will employ mixed qualitative methods, including: analysis of documents such as feedback forms, Knowledge Broker journal/diary, event attendance records, risk and issues logs and other relevant paperwork gathered as part of project delivery; observations of events/activities; and interviews with care home providers and staff, care home residents, residents' family members, and researchers who are involved in the project (both project design/delivery, and also attendance or involvement in project activities/events). Framework Analysis will be used to interpret the data collected; analysis will be strategic, by focusing on particular key areas of importance in the developing theory of how the ExCHANGE Collaboration might achieve change. RESULTS: The results of this study are expected to be published in 2022. DISCUSSION: This evaluation will investigate how successful the ExCHANGE Collaboration is perceived to be in achieving its aims, in what way, in which contexts, and how this may differ for those involved. It will do this by testing an initial programme theory about how the collaboration works, for whom, under which circumstances, and in what way. Findings will be shared through written publication, an end of project learning event for those involved/interested in the project, and a lay summary to be made publically available.

Back to basics: the development of a simple, homogenous, two-component dry-powder inhaler formulation for the delivery of budesonide using miscible vinyl polymers.

It was hypothesised that formulating a dry-powder inhaler (DPI) using a refined, smooth grade of lactose, without fines and a polymer coated drug microparticle should produce an homogeneous formulation in which aerosolization behaviour could be modified. Hence, the aim of this study was to develop a simple two component polymer coated-budesonide/lactose blend in which the drug microparticle adhesive forces could be optimised by modifying the drug coating in order to improve aerosolization from a DPI. Budesonide microparticles (1.83 +/- 0.03 microm) were coated with the vinyl polymers by adsorption and then spray-dried. The drug was blended with three different types of lactose, checked for uniformity of mixing and loaded into Pulvinal devices. The median volume particle size of all but one of the polymer coated microparticles remained below 4 microm after spray-drying and the content uniformity for all the blends >96%. Coating the budesonide with 0.01% poly(vinyl alcohol) increased the fine particle fraction (FPF) in the next generation impactor (NGI) from 29.1 +/- 0.7% to 52.8 +/- 1.0% and reduced the force of adhesion from 410 +/- 182 to 241 +/- 82 nN with smooth lactose. This illustrates that vinyl polymers could effectively modify adhesive interactions without the need for ternary components such as fines.

Multilayer PVA adsorption onto hydrophobic drug substrates to engineer drug-rich microparticles.

Despite the availability of numerous crystal engineering techniques, generating drug-rich microparticles with a predetermined size, morphology and crystallinity still represents a significant challenge. A microparticle manufacturing method has recently been developed that attempts to 'shield' the physicochemical properties of micronised drugs by the application of a microfine polymer coating. The aims of this study were to investigate the nature of the drug-polymer interactions and determine the effects of this manufacturing strategy upon release of the drug from the microparticles. The adsorption of poly(vinyl alcohol) (PVA) on the micronised hydrophobic drug surface was found to reach equilibrium between 23 and 27 h. The Freundlich isotherm model was shown to give the most accurate fit to the experimental data and thus multilayer adsorption was assumed. The adsorptive capacity (1/n) was specific to the substrate and PVA grade. An increase in the PVA (%) hydrolysis value caused 1/n to increase from 0.76 to 1.05 using budesonide and from 0.31 to 0.79 when betamethasone valerate (BMV) was used. Increasing the molecular weight of the adsorbing polymer caused a reduction in the strength of PVA-adsorbate interaction when budesonide was used as the substrate (from 0.76 to 0.59), whereas a three-fold increase (from 0.31 to 0.86) was achieved when the BMV substrate was employed. A proportion of the adsorbed polymer was shown to remain associated with the substrate during the spray-drying process and the polymer coating resulted in a significantly higher (p<0.05, ANOVA) amount of drug release in 60 min (ca. 100%) compared to budesonide alone.

Validation of a reverse-phase high performance liquid chromatographic method for concurrent assay of a weak base (salmeterol xinafoate) and a pharmacologically active steroid (fluticasone propionate).

The analysis of weakly basic drugs such as salmeterol xinafoate (SX) by reverse-phase liquid chromatography remains a problem, particularly when present in combination with other drugs such as steroids and weak acids. This study describes the validation of an assay for a weakly basic drug, salmeterol (SB), its weakly acidic counter-ion, 1-hydroxy-2-naphthoic acid (XA), and the neutral glucocorticoid, fluticasone propionate (FP) using a second-generation silica stationary phase (Inertsil ODS-2). The assay utilized an Inertsil ODS-2 base-deactivated 250 mm x 4.6mm, 5 microm HPLC column, with 75:25 methanol:0.6% aqueous ammonium acetate as the mobile phase. Under these near neutral conditions, SB demonstrated a good peak shape (tailing factor=1.21+/-0.02, n=85). The method provided a short analysis time: XA, t(R)=2.96 min; SB, t(R)=5.23 min and FP, t(R)=7.01 min. The assay displayed good sensitivity for both XA (LOD for SX=0.22 microgmL(-1)) and SB (LOD for SX=0.26 microgmL(-1)). The limit of detection for FP was 0.19 microgmL(-1). Neither of the drugs was found to interfere in the determination of the other and the assay accuracy (% recovery) was high (the recoveries were: 99.58+/-1.85% for XA, 99.49+/-1.88% for SB and 100.24+/-1.28% for FP). The assay reproducibility was determined with a mean coefficient of variance for the five calibration concentrations of XA=0.71+/-0.18%; SB=1.11+/-0.64% and FP=0.92+/-0.14%. Analysis of a pressurized metered dose inhaler formulation demonstrated recovery of the analytes that are within pharmacopoeial limits. It was shown that RP-HPLC was suitable for the high throughput analysis of the combination of SX and FP.

Tamoxifen decreases drug efflux from liposomes: relevance to its ability to reverse multidrug resistance in cancer cells?

Tamoxifen decreased the efflux of the fluorescent marker drug, chloroquine, from phosphatidylcholine liposomes. Tamoxifen is a known structural-mimic of cholesterol, which were both found to be similarly effective in preventing drug release from liposomes. This ability of tamoxifen and cholesterol to decrease drug efflux in a concentration-dependent manner is likely to arise from their known ability to decrease membrane fluidity both in liposomes and also in cancer cells. The possible importance of the ability of tamoxifen to inhibit drug efflux from liposomes in relation to its ability to reverse multidrug resistance in cancer patients caused by the efflux of cytotoxic therapeutic agents, is discussed.

High-resolution PET imaging and quantitation of pharmaceutical biodistributions in a small animal using avalanche photodiode detectors.

UNLABELLED: The feasibility of high-resolution PET using BGO-avalanche photodiode detectors for in vivo imaging and quantitation of the biodistribution of radiopharmaceuticals in small animals is demonstrated. A prototype PET camera consisting of two scanning arrays of eight EG&G C30994 solid-state scintillation detectors was used to simulate a 310-mm diameter dual-ring animal tomograph having a 130-mm port and three imaging slices, each about 3.5 mm thick. The spatial resolution (FWHM) is 3 mm or less, isotropic and uniform throughout the 120-mm diameter field of view. METHODS: Female Fischer 344/CRBL rats implanted with subcutaneous mammary adenocarcinoma tumors were injected with copper-tetrasulfophthalocyanine (CuPcS4), a potential sensitizer for the photodynamic therapy of cancer, labeled with 64Cu (T1/2 = 12.7 hr, beta +:19%). RESULTS: In spite of the low specific radioactivity of 64Cu and other inherent limitations, organs such as the liver, kidneys and the tumor could be resolved with sufficient detail for their separation and quantitation. Apart from the tumor, agreement was obtained between the biodistributions measured by PET and by scintillation counting. The discrepancy for the tumor measurement results from averaging the radioactivity over the entire tumor volume when, in fact, CuPcS4 does not completely penetrate the tumor. This incomplete penetration is noted on the PET images. CONCLUSIONS: PET based on avalanche photodiode detectors provides an accurate measurement of target organ and tumor tissue concentrations. These preliminary results demonstrate the potential of very high resolution PET for biodistribution studies in small animals.

Locally increased uptake of fluorine-18-fluorodeoxyglucose after intracavitary administration of iodine-131-labeled antibody for primary brain tumors.

UNLABELLED: After the intracavitary administration of 131I-labeled monoclonal antibody for treatment of primary brain tumors after surgical resection, a persistent rim of 18F-fluorodeoxyglucose (FDG) accumulation surrounding the cavity can be observed on PET. This rim, although it accumulates more FDG than adjacent normal brain tissue, is not necessarily associated with tumor. In our study, we examine the characteristics of the rim that indicate persistent tumor and tumor progression. METHODS: Sequential PET studies obtained after treatment in 10 patients were reviewed and the results correlated with dosimetry and post-treatment histologic diagnoses. RESULTS: The rim of FDG accumulation was seen on the first post-treatment scan obtained 1-3 mo after therapy and persisted unchanged over the 2-26 mo follow-up period. Pathologically, the nonmalignant rim was associated with marked increase of macrophage infiltrates. Nodularity of the rim was associated with tumor. CONCLUSION: Our study demonstrates that a rim of FDG accumulation is seen after intracavitary administration of 131I-labeled monoclonal antibody therapy independent of the presence of malignant disease. Malignant recurrence is suggested by the development of new nodularity in the rim of FDG accumulation.

The viscoelastic nature of mucus secretion.

The viscoelastic properties of mucus have been described together with the methods which can be used to evaluate such a rheologically complex system. Methods which do not take into account the viscoelastic nature of mucus have been criticized and it is concluded that only creep or oscillatory tests yield significant correlations with mucociliary clearance rates.

Hepatitis B screening in a northern Irish mental handicap institution: relevance to hepatitis B vaccination.

The current DHSS guidelines on immunisation against hepatitis B in mental handicap hospitals recommend vaccination for personnel at risk directly involved in patient care (who may have direct contact with patients or their body fluids) and for new admissions into institutions where the incidence is known to be high. We report a serological survey of hepatitis B markers in over 99% of the residents of a large mental handicap hospital. Of 720 patients tested only one carried hepatitis surface antigen. This patient was anti-HBe positive. Only 4.5% of the residents carried any hepatitis marker. These results do not suggest the need for a local vaccination programme for patients or staff.

The solubilization of progesterone by mixed bile salt-phospholipid sols.

The solubility of progesterone was determined in several different bile salt-phospholipid mixtures, and it is concluded that: (1) The solubility in unconjugated bile salts is greater than in the conjugated analogues, and the solubility in deoxycholate solutions is twice that in cholate solutions. (2) Substitution of hydroxyl groups in the 11 and 21 positions of progesterone increases solubility, whilst substitution in the 17-position decreases solubility in bile salt solutions. (3) Progesterone solubility in mixed bile salt solutions is proportional to the mole ratio of the surfactant mixture. (4) Sodium deoxycholate (SDC)-phospholipid sols show no such linear solubilizing properties; a minimum occurring at a mole ratio of SDC to phospholipid of 1 : 4. (5) There is a break in the solubility curve of progesterone in lysophosphatidycholine (LPC)/phosphatidylcholine (PC) mixtures at a mole ratio of 65 : 35 coincident with maximum viscosity. (6) Introduction of SDC into LPC/PC mixtures results in decreased progesterone solubility.

The use of lactose recrystallised from carbopol gels as a carrier for aerosolised salbutamol sulphate.

Lactose was crystallised either from Carbopol gel without stirring or from a constantly-stirred aqueous solution, to obtain lactose crystals designated as Carbo and control lactose, respectively. The Carbo lactose was shown to have a more regular shape with smoother surface as compared with the control lactose. These lactoses were fractionated by sieving to produce batches with different sizes before blending separately with salbutamol sulphate (SS, VMD 5.8 microm) in a ratio of 67.5:1 w/w using the same mixing procedure. SS dispersion and deaggregation were investigated using a 4-stage liquid impinger after aerosolisation at 28.3, 60.0 and 96.0 l/min via a Rotahaler. At all flow rates, the Carbo lactose produced significantly higher (ANOVA, P<0.01) emission of SS from the Rotahaler as compared with the control lactose of a similar size. The Carbo lactose also resulted in a significantly (P<0.05) higher fine particle fraction of SS than the control lactose. Moreover, drug emission from formulations containing the Carbo lactose was consistently more reproducible than those of the control lactose blends. In conclusion, the efficiency and reproducibility of drug delivery by dry powder inhalers can be improved using carrier particles of precisely defined morphological features.

Rheological evaluation of hog gastric mucin as a model mucus system.

The rheological evaluation of preparations containing 10, 15, 20, and 25% powdered hog gastric mucin was carried out over the 13-48 degrees temperature range using rotational and creep viscometry. The preparations were almost viscous, and no elastic behavior was demonstrated. The addition of borate ions frequently produced a slight decrease in viscosity. Tetracycline hydrochloride decreased the viscosity of the 10 and 25% materials, although the addition of this compound to fresh gastric and bronchial mucous gels markedly increased viscosity. The model is, therefore, only suitable in limited circumstances as a basis for the evaluation of the effect of drugs on gastric mucus.

Correlations between physical and drug release characteristics of polyethylene glycol suppositories.

The mechanical strength and elastic moduli of blocks of polyethylene glycol with a range of molecular weights were determined. A rotating-basket dissolution test was used to measure the release characteristics of prednisolone from similar blocks. The effects of blending bases of different molecular weight and of the addition of water also were determined. Linear relationships were found for the mechanical strength, molecular weight, and release rate, but no simple relationship could be observed for the elastic moduli.

Lactose as a carrier in dry powder formulations: the influence of surface characteristics on drug delivery.

The aim of the study was to investigate the interdependence of carrier particle size, surface treatment of the carrier, and inclusion of fines on the drug delivery from dry power inhaler formulations. Two size fractions (< 63 and 63-90 microm) of alpha-lactose monohydrate were subjected to treatment with 95% (v/v) ethanol to introduce small asperities or cavities onto the otherwise smooth surface without substantially changing the particle shape. After blending with albuterol sulfate [ALB; volume median diameter (VMD), 1.9 microm; geometric standard deviation (GSD), 1.5], the solvent-treated lactose produced a fine particle fraction (FPF; < 6.18 microm) and dispersibility of the drug that was significantly (ANOVA p < 0.01) lower than that which resulted from formulations containing untreated lactose of a similar size fraction, after aerosolization at 60 L min(-1) via a Rotahaler. The two size fractions of the treated lactose resulted in similar deposition profiles of ALB. The effects of such surface asperities or cavities of lactose were offset by introducing a small amount (5% w/w) of smaller-sized lactose (5-10 microm) to the powder formulations. The fine lactose increased the FPF and dispersibility of ALB to such a level that all lactose batches, regardless of particle size or whether solvent treated, produced a similar fraction of aerosolized ALB. The inclusion of recrystallized needle lactose (5-15 microm) was superior to micronized lactose in improving the aerosolization of ALB. The findings of this study indicate that the presence and characteristics of the finer fraction of lactose carrier particles dominate over the particle size and surface smoothness of the carrier particles in determining dispersion and deaggregation of drugs from dry powder formulations for inhalation.

Use of alveolar cell monolayers of varying electrical resistance to measure pulmonary peptide transport.

The apparent permeability coefficient (P(app)) of two fluorescently tagged model hydrophilic peptides, acXASNH(2) and acXAS(GAS)(7)NH(2), and (14)C-mannitol across monolayers of cultured rat alveolar epithelial cells of varying transepithelial electrical resistance (TER) has been examined. In line with their design features, the peptides were not degraded under the conditions of the test. Furthermore, no concentration dependence of transport of the tripeptide acXASNH(2) was observed over the concentration range studied, nor was any directional transport seen for either of the model peptides, indicating that under the conditions of the test they were not substrates for any transporters or efflux pumps. From the hydrophilic nature of the peptides (as assessed by their log P), and their inverse dependence of transport with molecular weight and TER, it was assumed that the peptides were transported across the cell monolayer passively via the paracellular route. The observed P(app) for the transport of (14)C-mannitol and the peptides across rat alveolar epithelial cell monolayers were found to be inversely (though not linearly) related to the measured TER and could be well-modeled assuming the presence of two populations of "pores" in the cell monolayer, namely, cylindrical pores of diameter 1.5 nm and large pores of diameter 20 nm. The relative populations of the two types of pores varied with the TER of the monolayer, with the number of large pores decreasing with an increase in TER (and the number of small pores taken as fixed). These results suggest that if the cell monolayer is well characterized with respect to the passage of a range of probe molecules across monolayers of varying electrical resistance, it should be possible to predict the P(app) of any hydrophilic peptide or drug crossing the membrane by the paracellular route at any desired TER using a monolayer of any electrical resistance, above a minimum value.

The migration of bacteria through gels in the presence of IUCD monofilament tails.

An in vitro model was developed to investigate the migration of a variety of bacteria of different characteristics through a gel system in the presence or absence of a wide range of polymer monofilament threads. The bacteria were unable to migrate through the gel from the point of inoculation in the absence of a solid substrate. Migration occurred along all thread types tested, including those used as IUCD marker tails and the extent of bacterial migration appeared to be determined primarily by the motility of the microorganisms. The implications of these findings in relation to the development of pelvic infections in IUCD wearers is discussed.

Dry powder inhalers: the influence of device resistance and powder formulation on drug and lactose deposition in vitro.

It is a principal in the formulation of a dry powder aerosol that the device should enable a high fine particle fraction (FPF) of drug to be delivered to the lung whilst any carrier, such as lactose, should remain in the upper airways. Both the device and the dry powder formulation itself contribute to the resultant FPF and few studies have considered the deposition of lactose carrier. It was the purpose of this study to determine the effect of the resistance of the device and the influence of powder formulation on the deposition of drug and carrier. Measurement of the pressure drop across the devices investigated in this study showed that the two types of Inhalator Ingelheim had the highest resistance, whilst lower pressure drops were found across the Diskhaler, Cyclohaler and Accuhaler devices. The lowest pressure drops were measured across the Rotahaler and Spinhaler devices. Employing Rotacaps 400 capsules as the formulated salbutamol product, the FPF of drug was greater from the high resistance devices, being in the order Inhalators Ingelheim>Cyclohaler>Rotahaler=Spinhaler. However, the Diskhaler, employing its own developed formulation, produced the highest FPF, approximately twice that from the Accuhaler. There was no statistical difference between the FPF of salbutamol (approximately 20% nominal dose) from the Rotacaps formulation when aerosolised using high resistance devices (Inhalators Ingelheim) operated at 30 l min-1, a medium resistance device (Cyclohaler) operated at 60 l min-1 and low resistance devices (Spinhaler and Rotahaler) operated at a flow-rate of 90 l min-1. The Ventolin Diskhaler using its own formulation operated at 60 l min-1 gave a FPF of 40.33%, but the FPF obtained was sensitive to flow, being only 25.65% of the nominal dose at 30 l min-1. Whereas no lactose was found in the FPF from the Accuhaler operated at 60 l min-1, 100, 400 and 3500 microg were obtained from the Diskhaler, Rotacaps and micronised lactose formulation, respectively, when operated at the same flow-rate. An in-house formulation comprising salbutamol sulphate blended with micronised lactose in a weight ratio of 1:67.5 and aerosolised from a Cyclohaler produced a similar FPF to the Diskhaler at 60 l min-1. When air flow was reduced to 30 l min-1, the FPF from the in-house formulation was reduced considerably less than that from the Diskhaler formulation.

Effects of molecular weight of polyvinylpyrrolidone on the glass transition and crystallization of co-lyophilized sucrose.

The purpose of the present study was to investigate the effects of molecular weight (MW) of polyvinylpyrrolidone (PVP) on glass transition and crystallization of sucrose. Thus, sucrose was co-lyophilized with 2.5 and 5.0% w/w PVP of different molecular weights, which were characterized using gel permeation chromatography. Freeze drying was carried out for 48 h at a shelf temperature of -40 degrees C and a pressure of about 36 Pa. The samples were then dried in a vacuum oven at 24 degrees C for 12 h before drying for a further 12 h at 40 degrees C. Differential scanning calorimetry (DSC) was employed to measure the glass transition temperature (Tg), dynamic crystallization temperature (Tc) and isothermal crystallization induction time (tc) at 85 degrees C of sucrose. Isothermal water vapour sorption of each sample was also measured at different relative humidities. Tg values of sucrose varied from 48.3+/-0.8 degrees C for freeze-dried (FD) sucrose alone to 58.8+/-0.8 degrees C for the mixture containing 5.0% PVP of nominal MW 300 K. PVP increased sucrose T(g) significantly (ANOVA P<0.05). Although there was no significant difference (P>0.05) in Tg of the mixtures containing 2.5% w/w PVP of different MW, samples with 5.0% PVP of MW 300 K produced a significantly higher (P<0.05) Tg than the other mixtures. All mixtures were shown to possess higher (P<0.01) Tc than FD sucrose alone, which exhibited a T(c) of approximately 85 degrees C. PVP of MW 300 K consistently induced a significantly (P<0.05) higher Tc of sucrose than PVP of smaller MW. Increasing PVP concentration from 2.5 to 5.0% also resulted in a substantial increase in sucrose Tc. Using isothermal water vapour absorption, sucrose tc was found to increase up to over 10 times when it was co-lyophilized with 2.5% PVP, the actual value of tc being dependent upon the MW of the PVP. For example, PVP of MW 300 K resulted in a sucrose tc at 85 degrees C (89.1-95.6 min), which was approximately seven times higher than that of 2.5% PVP of MW 24 or 40 K. A longer tc of sucrose was also observed for mixtures containing PVP of MW 300 K than when sucrose was mixed with PVP of smaller MW. Thus the effect of PVP on sucrose Tg, Tc and tc was found to be dependent upon MW. PVP of higher MW was more efficient in inhibiting sucrose crystallization and by stabilizing glassy structures of the sugar, these polymers may improve the stability of co-lyophilized proteins and peptides.