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Infections caused by opportunistic fungal pathogens are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about the extent to which susceptible patients acquire infection from drug resistant genotypes in the environment. Here, we present a population genomic analysis of the mouldAspergillus fumigatusfrom across the United Kingdom and Republic of Ireland. First, we show occurrences where azole resistant isolates of near identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Second, we find that the fungus is structured into two clades (‘A’ and ‘B’) with little interclade recombination and the majority of environmental azole resistance genetically clustered inside Clade A. Genome-scans show the impact of selective sweeps across multiple regions of the genome. These signatures of positive selection are seen in regions containing canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, whilst other regions under selection have no defined function. Phenotyping identified genes in these regions that could act as modifiers of resistance showing the utility of reverse genetic approaches to dissect the complex genomic architecture of fungal drug resistance. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Standardization of Aspergillus polymerase chain reaction (PCR) protocols has progressed, and analytical validity of blood-based assays has been formally established. It remains necessary to consider how the tests can be used in practice to maximize clinical utility. To determine the optimal diagnostic strategies and influence on patient management, several factors require consideration, including the patient population, incidence of invasive aspergillosis (and other fungal disease), and the local antifungal prescribing policy. Technical issues such as specimen type, ease of sampling, frequency of testing, access to testing centers, and time to reporting will also influence the use of PCR in clinical practice. Interpretation of all diagnostic tests is dependent on the clinical context and molecular assays are no exception, but with the proposal to incorporate Aspergillus PCR into the second revision of the consensus guidelines for defining invasive fungal disease the acceptance and understanding of molecular tests should improve.

The galactomannan enzyme immunoassay (GM-EIA) is widely utilized for the diagnosis of invasive aspergillosis (IA). There is inconsistent reproducibility of results between centers when the assay is processed manually. Automation of EIAs can reduce variation. This study investigated the semiautomation of the GM-EIA on the DS2 (Dynex) platform in the following three stages: (i) DS2 GM-EIA method validation with experimental samples, (ii) DS2 retesting of case-defined clinical samples, and (iii) a 12-month audit of DS2 GM-EIA performance. In stage i, Bland-Altman analysis demonstrated a reduced variance between optical density index (ODI) values for samples processed on two DS2 platforms (mean difference, −0.02; limits of agreement [LOA], −0.19 to 0.14) compared with the variance between samples processed manually and on a DS2 platform (mean difference, 0.02; LOA, −0.25 to 0.3). In stage ii, 100% (14/14 samples) qualitative agreement was observed for serum samples from patients with IA, with no significant change in the ODI values when samples were processed on the DS2 platform. A significant decrease in ODI values was observed for control serum samples on the DS2 platform (difference, 0.01; P = 0.042). In stage iii, a significant reduction in the frequency of equivocal results, from 5.56% (136/2,443 samples) to 1.56% (15/961 samples), was observed after DS2 automation (difference, 4.0%; 95% confidence interval [CI], 2.7 to 5.2%; P < 0.01), with an equivalent increase in negative results. This study demonstrates that GM-EIA automation may reduce intersite variability. Automation does not have an impact on the repeatability of truly positive results but contributes to a reduction in false-positive (equivocal) GM-EIA results, reducing the need to retest a significant proportion of samples.

Diagnosis of invasive aspergillosis (IA) remains challenging. With a relatively low incidence of disease, the use of expensive empirical antifungal therapy exposes many patients to unnecessary toxicity. Diagnosis places emphasis on specific but temporal radiological evidence. Circulating biomarker diagnosis has shown potential, but assays show variable performance, take several hours to perform, and require a degree of technical ability. A novel and simple lateral-flow device (LFD) using monoclonal antibody JF5, which targets an extracellular glycoprotein, has been developed and potentially removes any technical requirements, reducing processing time considerably. In this study, we evaluate the performance of this LFD compared to real-time PCR (targeting the 28S rRNA gene) and galactomannan (GM) detection when testing serum from a European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG)-defined hematological population. In a proven/probable-IA population versus a no-IA population, the LFD performance was comparable to that of both PCR and galactomannan enzyme immunoassay. Specificity (98.0%) was similar to that of PCR (96.6%) and slightly superior to that of GM (91.5%). Sensitivity (81.8%) was inferior to that of PCR (95.5%) but better than that of GM (77.3%). In combination with PCR, it provided both 100% sensitivity and 100% specificity. The LFD permits rapid testing of easily obtainable specimens, to be used as an adjunct test, before confirmation by other investigations. Its simplicity provides centers without specialist diagnostics with a test with clinical performance superior to that of classical microbiological approaches and results that can be used to direct antifungal management. In summary, microbiological diagnosis of IA is difficult and options are limited, with variable performance. An LFD assay targeting a novel specific biomarker has been developed, one which is methodologically simple and provides good clinical performance, particularly if combined with PCR.

PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 μl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (≥3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of

There are conflicting reports on the agreement between the Clinical and Laboratory Standards Institute disk diffusion (M44-A) and reference broth microdilution (M27-A) methods for determination of antifungal susceptibility of yeasts. The antifungal susceptibility of 541 yeasts, the majority of which were from the oral cavity, was determined using these two methods and the accuracy of the disk diffusion method assessed for clinical testing of various Candida species. Of the strains tested, Candida albicans predominated (390 out of 541). The classification of susceptibility determined by the disk diffusion method was largely in concordance with that obtained using the broth dilution method, regardless of species within Candida genus. The overall observed agreement between these two methods was 94.7% for fluconazole and 96.7% for voriconazole was with a 'very major' discrepancy level of 1.5% and 1.7% respectively. This study demonstrates a strong agreement of the simple disk diffusion method with the more labour intensive 'gold standard' broth microdilution method. These findings would support the use of the disk diffusion method in a routine mycology service.

Endocarditis secondary to Aspergillus niger has not been described in a leukaemic patient. We describe a case of A. niger endocarditis in a patient with acute myeloid leukaemia and refractory fever. The microbiological cause of his endocarditis was initially misdiagnosed because he fulfilled the Duke criteria for enterococcal endocarditis. A polymerase chain reaction test utilizing pan-fungal primers detected a product from an Aspergillus sp. The DNA was subsequently sequenced and was found to have 100% homology with A. niger. A postmortem revealed fungal endocarditis secondary to disseminated aspergillosis, without evidence of bacterial endocarditis. The patient was found to have a lung aspergilloma that was possibly occupationally acquired, and may have been long standing.

Invasive fungal disease is a significant cause of morbidity and mortality in the immunocompromised patient. Early and accurate diagnosis is essential to improve outcome. This article explores non-culture based methods as aids to diagnosis as well as a means to risk stratify patients in order to determine the need for targeted therapy.

Foodborne illness is a major public health concern. The largest number of foodborne illness cases attributed to poultry and poultry products are caused by paratyphoid serotypes of Salmonella and by Campylobacter jejuni. The effective prevention of foodborne disease requires an understanding that contamination can be introduced into foods at numerous points along the food chain. Since multiple entry points exist for foodborne pathogens, multifaceted intervention approaches are required to successfully control contamination of poultry during the various phases of the growth period and processing procedure of broiler chickens. Strategies during the grow-out period (the period during which day-old chicks are raised to six- to seven-week-old broiler chickens) include sanitation, biosecurity, vaccine and drug therapy, and biological control procedures, such as those aimed at preventing colonisation. There are also many critical control points identified in the processing plant which reduce contamination. These include temperature controls (washer and product), chemical interventions, water replacements and counter-flow technology in the scalder and chiller, and equipment maintenance. Transportation and food handling at retail outlets and by the consumer (i.e., storage at the proper temperature and adequate cooking) are the final critical control points in the farm-to-table continuum. It is important to apply risk reduction strategies throughout the food chain. These include: easing the development and implementation of voluntary animal production 'best management practices', implementing in-plant hazard analysis and critical control point systems, developing effective transportation and refrigeration standards, working to facilitate adoption of the model Food Code in all States and providing educational materials and support for public health activities nationwide.

SUMMARYWe investigated an outbreak of 396Salmonella entericaserotype I 4,5,12:i:- infections to determine the source. After 7 weeks of extensive hypothesis-generation interviews, no refined hypothesis was formed. Nevertheless, a case-control study was initiated. Subsequently, an iterative hypothesis-generation approach used by a single interviewing team identified brand A not-ready-to-eat frozen pot pies as a likely vehicle. The case-control study, modified to assess this new hypothesis, along with product testing indicated that the turkey variety of pot pies was responsible. Review of product labels identified inconsistent language regarding preparation, and the cooking instructions included undefined microwave wattage categories. Surveys found that most patients did not follow the product's cooking instructions and did not know their oven's wattage. The manufacturer voluntarily recalled pot pies and improved the product's cooking instructions. This investigation highlights the value of careful hypothesis-generation and the risks posed by frozen not-ready-to-eat microwavable foods.

We study the cosmology of the Supersymmetric Standard Model augmented by a gauge singlet to solve the $\mu$-problem and describe the evolution of the domain walls which are created during electroweak symmetry breaking due to the discrete $Z_{3}$ symmetry in this model. The usual assumption, that non-renormalizable terms induced by gravity (which explicitly break this symmetry) may cause the walls to collapse on a cosmologically safe timescale, is reconsidered. Such terms are constrained by considerations of primordial nucleosynthesis, and also by the fact that by not respecting the $Z_{3}$ symmetry they induce divergences which destabilise the hierarchy and reintroduce the $\mu$--problem. We find that, even when the K\"ahler potential is `non-minimal' (i.e. when the hidden sector couples directly to the visible) the model is either ruled out cosmologically or suffers from a naturalness problem., Comment: 17 pages, plain LaTeX, figures as uu file

Sets up the consistency conditions for super Yang-Mills coupled to the Wess-Zumino multiplet in the Wess-Zumino gauge with the auxiliary fields eliminated, after closing the algebra using the formulation of Batalin and Vilkovisky (1983). The resulting cohomology is then solved with the method of spectral sequences.

The nilpotent symmetry operator for the full superconformal symmetry of N=4 super Yang-Mills is constructed using the method of Batalin and Vilkovisky (1977). Spectral sequences are then used to prove that the cohomology of this operator is trivial, and so the theory has no possible anomalies. This shows that all symmetries are preserved after quantization, and so there is no conformal anomaly and the theory is thus finite to all orders.

Raman microprobe studies of pulsed laser damaged TiO2 films deposited using three different methods are reported. Phase transformation and redeposition of coating materials were observed in selected regions of amorphous films deposited by ion beam sputtering and electron beam evaporation. Preferential removal of a specific phase or transformation to a second phase were observed in reactively sputtered films. Some damage sites exhibited regions of stress heterogeneity which can be explained in terms of the return electron stream model of plasma/target interaction and rapid quenching.

Aims: To investigate the impact of routine use of biomarkers for diagnosing fungal infection within a care pathway on antifungal usage and clinical outcomes. Methods: A cohort of high-risk haematology and stem cell transplant patients was entered into a neutropenic care pathway in which targeted diagnostic testing replaced empiric antifungal treatment. Patients were screened twice a week by PCR and antigen testing during fever or when chronic graft versus host disease was present and were followed-up for a minimum of 1 year. Results: No excess morbidity or mortality was seen in patients in whom empiric antifungal treatment was withheld, and there were substantial savings in antifungal drug expenditure. Conclusions: The introduction of a comprehensive diagnostic surveillance strategy to exclude invasive fungal infection in high-risk patients with haematological malignancy and those undergoing transplantation can result in improvements in clinical management. There are also potential additional benefits of improved patient survival, decreased morbidity and decreased hospital stay.

The polymerase chain reaction (PCR) is a common tool of molecular biology and is utilized in the field of medical diagnostics to identify genetic disorders and the causative agents of human infections. PCR is also used for typing microorganisms, thus playing an important role in both epidemiology and phylogenetics. The PCR method is a starting point that when used together with cloning is influential in the sequencing of novel genes and genomes.

Foodborne illness is a major public health concern. The largest number of foodborne illness cases attributed to poultry and poultry products are caused by paratyphoid serotypes of Salmonella and by Campylobacter jejuni. The effective prevention of foodborne disease requires an understanding that contamination can be introduced into foods at numerous points along the food chain. Since multiple entry points exist for foodborne pathogens, multifaceted intervention approaches are required to successfully control contamination of poultry during the various phases of the growth period and processing procedure of broiler chickens. Strategies during the grow-out period (the period during which day-old chicks are raised to six- to seven-week-old broiler chickens) include sanitation, biosecurity, vaccine and drug therapy, and biological control procedures, such as those aimed at preventing colonisation. There are also many critical control points identified in the processing plant which reduce contamination. These include temperature controls (washer and product), chemical interventions, water replacements and counter-flow technology in the scalder and chiller, and equipment maintenance. Transportation and food handling at retail outlets and by the consumer (i.e., storage at the proper temperature and adequate cooking) are the final critical control points in the farm-to-table continuum. It is important to apply risk reduction strategies throughout the food chain. These include: easing the development and implementation of voluntary animal production 'best management practices', implementing in-plant hazard analysis and critical control point systems, developing effective transportation and refrigeration standards, working to facilitate adoption of the model Food Code in all States and providing educational materials and support for public health activities nationwide.

We discuss the discovery reach of CERN LEP 2 for the Higgs sector of a general extension of the MSSM including a single gauge singlet field. This change introduces a new quartic Higgs boson self-coupling which can increase the masses of the {ital CP}-even states, and also allows mixing between singlet and nonsinglet states which can reduce the couplings of the mass eigenstates to the {ital Z}. The lightest {ital CP}-even Higgs boson is bounded by a parameter {Lambda} which takes a maximum value {Lambda}{sub max}{approx_equal} 136{endash}146 GeV for a top quark mass of 150{endash}195 GeV. We generalize the discussion of the bound to include the entire {ital CP}-even spectrum and show how experiment may exclude values of {Lambda} smaller than some {Lambda}{sub min}. {ital CP}-even Higgs boson searches at LEP 2 will be able to exclude {Lambda}{sub min}{approx_equal} 81{endash}105 GeV, depending on the machine parameters. We also present exclusion plots in the {ital m}{sub {ital A}}-tan{beta} plane, based on an analysis of {ital CP}-even, {ital CP}-odd, and charged Higgs boson production processes at LEP 2. {copyright} {ital 1996 The American Physical Society.}

Previous attempts to induce mucosal antibodies in rhesus monkeys by enteric immunization have resulted in only modest and short-lived responses, dominated by immunoglobulin M (IgM) antibodies in the plasma. In this study, two groups of rhesus monkeys were immunized intranasally three times at 2-week intervals with a bacterial protein antigen (AgI/II) either chemically coupled to or mixed with the B subunit of cholera toxin (CT), a known potent mucosal immunogen and carrier for other immunogens. Cells secreting antibodies, predominantly of the IgA isotype, to AgI/II and to CT were detected in the peripheral blood 1 week after each immunization, indicating the dissemination of IgA-secreting precursor cells through the mucosal immune system. IgG and, to a lesser extent, IgA antibodies to both proteins were induced in the plasma commencing after the second immunization. Plasma IgE concentrations and IgE antibody levels were not consistently raised during the immunization period. IgA antibodies were found in nasal and vaginal washes. Nasal IgG but not IgA antibodies showed a significant positive correlation with plasma IgG antibody levels, suggesting that they were largely derived by transudation from the circulation. Analysis of the molecular form of vaginal IgA indicated that both secretory and monomeric forms of IgA were present in various proportions. Furthermore, neither IgG nor IgA antibodies in vaginal washes were correlated with plasma antibody responses, suggesting the contribution of locally synthesized antibodies of both isotypes. Comparison of the responses between the two groups of animals showed only sporadic significant differences, indicating that intranasal immunization with AgI/II either coupled to or mixed with the B subunit of CT was equally effective at inducing generalized IgA antibody responses in the mucosal immune system and predominantly IgG antibodies in the plasma.

We present full renormalisation group equations for the MSSM with R-parity violation, including all soft supersymmetry breaking terms. The inclusion of dimensionless R-parity violating couplings can generate many possible low energy effects through the generation of off-diagonal soft masses violating lepton and quark flavour, and through the generation of lepton-Higgs mixing. We discuss the relation between the weak and unification scale R-parity violation and study the effects on neutrino mass generation and $\mu\to e\gamma$., Comment: 41 pages plain LaTeX, 15 figures all uuencoded and gzipped (typos fixed and trivial changes ready for Phys. Rev. D)

We perform a detailed analysis of the next-to-minimal supersymmetric standard model (NMSSM), imposing the constraints of two-loop gauge coupling unification, universal soft supersymmetry breaking and the correct pattern of electroweak symmetry breaking. We compare our results with those for the minimal supersymmetric standard model (MSSM) using closely related techniques and, as far as possible, a common set of input and output variables. In general, in the constrained NMSSM, there are much stronger correlations between parameters than in the constrained MSSM, and we map out the allowed parameter space. We also give a detailed discussion of how to resolve the two models experimentally, concentrating primarily on the prospects at LEPII., Comment: 76 pages, latex, no macros, uuencoded figures included separately. This version (to appear in Phys. Rev. D) contains various minor changes

Establishing an exclusive minimally invasive surgery suite requires a commitment from management, administration, staff, and physicians. A specialized, dedicated staff becomes devoted to perfecting the procedures. There are many rewards associated with this obligation, the most rewarding being improvement in total quality patient care.

Results from a matrix study of the effects of fabrication technique (and attending subsurface damage) on the properties of ion beam milled fused silica surfaces are presented. Measured properties include surface and subsurface scatter, surface absorption, and laser damage threshold. Correlations between subsurface damage induced during fabrication and notable surface properties are identified. A figure of merit is used to identify the fabrication techniques best suited for use with ion beam milling and figuring.

Results of a diagnostic matrix designed to characterize ion beam milled optical surfaces prepared using a selected set of polishing parameters are presented and discussed.

The dynamic elastic moduli were measured on macroscopic models simulating two-phase microstructures where the second phase was present as a continuous, isotropic network. Using open-pore polymer foams with higher-moduli, infiltrating matrices, the composite moduli were found to be linear with volume fraction of the network phase. Variations of network cell size or surface area indicated that the moduli increased with increasing interfacial area, but only when interphase wetting was present and matrix contraction upon solidification occurred. Metallic foams with a lower moduli matrix produced moduli that were compatible with existing bounding theories for composites.

This investigation concerns the comparison of two- and three-dimensional data obtained on a cellular material. By quantitative micrography techniques and spatial measurements, the cellular structure corresponds most closely with the shape of pentagonal dodecahedra, twelve-faced cells having five edges per face. The cell volumes have a normal distribution. The areal distributions of planar sections for various shapes of polyhedra were taken from existing literature. The measured volume distributions from this study were then applied by numerical computer calculations to create a transformation which carried the distribution of volumes (three-dimensional) to the expected distribution of planar areas (two-dimensional). The results of the expected and measured areal data agreed well for assumed complex polyhedral symmetry such as pentagonal dodecahedra, and unsatisfactorily for spherical symmetry. These results demonstrate that the pentagonal dodecahedron is a measurable prototype of cell in grain shapes.

Young male rats fed an atherogenic diet for 24 to 26 days developed magnesium deficiency. The renal lesions, calcium deposition in tubular lumens in the outer zona intermedia, are morphologically consistent with those seen in animals fed magnesium-free diets. It was necessary to feed 8 to 16 times the normal requirement of magnesium to prevent completely these lesions. The limitations in the pathogenesis of these renal lesions are discussed. The addition of 2 mg. per cent of thyroxine to the diet markedly lowered or abolished deposition of calcium in the kidney; it also lowered atherogenesis to a minimum but did not abolish it. Early lesions of atherosclerosis of the cardiac valves and of the aorta were observed. These were more prominent in animals on a 10 per cent than in those on a 20 per cent protein diet. The atherosclerosis was diminished but not abolished by exceedingly high dietary levels of magnesium in the 10 per cent protein group. Such a decrease was not noted in the 20 per cent protein group. There did not appear to be significant variation in the morphology of the fatty livers in and between the various groups and subgroups as judged by staining with Sudan IV.

The concept of "missing" is defined as failure to diagnose cancer before it has spread beyond the organ of origin. Data are presented for the 11 sites at which cancer occurs most frequently in the United States. There is a clear association between the survival of patients with cancer of a particular site and the percentage of cancers of that site diagnosed in a localized stage. However, the reasons for variation in the proportion of cases discovered while localized, which varies from 79% for uterine corpus to 12% for pancreas, appear to be varied and complex. Available data suggest that the nature of the tumor-host relationship and metastatic potential of the neoplasm are probably more important than alertness on the part of the patient and the physician, and probably have a more profound influence on stage of disease at diagnosis than currently available screening techniques.

A survey of prescribed medicines was carried out in 192 homes, broadly representative of households in England and Wales. The average home had 3.2 containers of such medicines; 56 per cent were in current use, 16 per cent in occasional use, and 28 per cent were never used. One fifth of all oral antibiotics found in the study were wasted. A rough estimate suggests that pound23 million of prescription products (5.6 per cent of the total) are wasted annually in England and Wales.

Three experiments were conducted to examine the effects of 35% N2 O (nitrous oxide) on human memory and auditory perception. In Experiment I, dichotic listening performance was found to be impaired. Experiment II used the same technique but was controlled for attenuation of sound transmission in the middle ear. No impairment was found. The perceptual effect found in Experiment I was peripheral, not central, and N2O did not impair short-term memory (STM). Experiment III used one-trial free recall of a word list. The shapes of the serial position curves were interpreted as indicating that N2O impairs long-term memory (LTM) but not STM. Experiment III provided no evidence, using cued recall, that the LTM deficit was due to impaired retrieval. Comparing these results with those for compressed air led to the conclusion that both N2O and hyperbaric nitrogen display an identical pattern of effects. A reason for the decrement found in some N2O STM studies may have been confounding the measurement of STM with that of LTM.