Itraconazole resistance in Aspergillus fumigatus

Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.     

In vitro activity of the echinocandin antifungal agent LY303,366 in comparison with itraconazole and amphotericin B against Aspergillus spp

LY303,366 (LY) is a novel derivative of the echinocandin class of antifungal agents. The in vitro activities of LY, itraconazole (ITZ), and amphotericin B (AMB) were assessed against 60 Aspergillus isolates, including 35 isolates of A. fumigatus, eight isolates of A. terreus, eight isolates of A. flavus, eight isolates of A. niger and one isolate of A. nidulans. Four A. fumigatus isolates were resistant to ITZ. Susceptibility testing for all drugs was performed with a broth microdilution procedure. LY was tested in two media: antibiotic medium 3 (AM3) and Casitone with 2% glucose (CAS) with an inoculum of 2 x 10(3) spores/ml. ITZ and AMB were tested in RPMI 1640 with 2% glucose with an inoculum of 1 x 10(6) spores/ml. All tests were incubated at 37 degrees C for 48 h. A novel end point was used to determine a minimal effective concentration (MEC) for LY, i. e., almost complete inhibition of growth save a few tiny spherical colonies attached to the microplate. MICs were measured for ITZ and AMB with a no-growth end point. Ranges and geometric mean (GM) MECs were from 0.0018 to >0.5 and 0.0039 mg/liter and from 0.0018 to >0.5 and 0.008 mg/liter for LY in AM3 and LY in CAS, respectively. Differences between species were apparent, with A. flavus being significantly less susceptible to LY than any other species tested with both media (P 16 and 0.7 mg/liter for ITZ and from 0.25 to 16 and 1.78 mg/liter for AMB. Minimal fungicidal concentrations (MFCs) were also determined for all drugs. GM MFCs were 0.018, 0.09, 19.76, and 12.64 mg/liter for LY in AM3, LY in CAS, ITZ, and AMB, respectively. LY in AM3 and LY in CAS were fungicidal for 86.7 and 68% of isolates, respectively (98% killing). In comparison, ITZ and AMB were fungicidal for 35 and 70% of isolates, respectively (99.99% killing). A reproducibility study was performed on 20% of the isolates. For 12 isolates retested, the MEC or MIC was the same or was within 1 dilution of the original value for 11, 11, 10, and 9 isolates for LY in AM3, LY in CAS, ITZ, and AMB, respectively. In conclusion, LY seems to be a promising antifungal agent with excellent in vitro activity against Aspergillus spp.     

Ulcerative colitis and coexisting colorectal cancer: recurrence rate after restorative proctocolectomy

BACKGROUND: The association between mucosal ulcerative colitis (MUC) and adenocarcinoma is well established. METHODS: Records of patients who had undergone restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) from 1983 through 1992 were examined. Of these, 604 had MUC and 27 (4.3%) had MUC with coexisting cancer. Patients were surveyed annually for recurrent disease. Pouch function and quality of life were evaluated with a questionnaire and physical examination. RESULTS: The duration of disease was longer (p = 0.001) in patients with cancer (16.1 +/- 8.0 years) than in those without cancer (9.1 +/- 7.1 years), although the mean age at diagnosis of MUC was the same. Of the 27 patients, 20 had colon cancer and seven had rectal cancer. Multicentricity was found in seven (25.9%) patients. Using the TNM staging classification, 14 patients (51.8%) had stage 1 cancer, eight (29.6%) had stage 2, four (14.8%) had stage 3, and one (3.8%) had stage 4. The patient with stage 4 cancer died 5 months after surgery and was excluded from the follow-up analysis. During a mean follow-up time of 4.3 +/- 2.6 years, cancer recurred in two of the remaining 26 patients (7.7%). In one patient, a local recurrence was found 8 months after surgery, and distant metastases were found in the other patient 35 months after surgery. Both recurrences were in patients with colon cancer. Two of the 26 patients died; one death was related to cancer recurrence (3.8%). Pouch function is good to excellent in all surviving patients. CONCLUSIONS: Restorative proctocolectomy for patients with MUC and coexisting colorectal cancer can be performed with a favorable prognosis and function. It is appropriate for curative intent, given that an adequate margin without tumor is obtained.     

Isolation of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Ground Beef Using Modified Rainbow Agar and Post-Immunomagnetic Separation Acid Treatment

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.     

Enhanced in vivo Optical Imaging of the Inflammatory Response to Acute Liver Injury in C57BL/6 Mice Using a Highly Bright Near-Infrared BODIPY Dye

Delving deeper is possible in whole-body in vivo imaging using a super-bright membrane-targeting BODIPY dye ( BD ). The dye was used to monitor homing of ex vivo fluorescently labelled neutrophils to an injured liver of dark-pigmented C57BL/6 mice. In vivo imaging system (IVIS) data conclusively showed an enhanced signal intensity and a higher signal-to-noise ratio in mice receiving neutrophils labelled with the BD dye relative to those labelled with a gold standard dye at 2 h post in vivo administration of fluorescently labelled cells. Fluorescence-activated cell sorting (FACS) confirmed that BD is nontoxic, and an exceptional cell labelling dye that opens up precision deep-organ in vivo imaging of inflammation in mice routinely used for biomedical research. The origin of enhanced performance is identified with the molecular structure and the distinct localisation of the dye within cells that enable remarkable changes in its optical parameters.     

The determination of surface tension at elevated temperatures by drop image analysis

The advantages of image analysis as applied to surface tension measurements by the sessile drop technique are discussed. It is demonstrated that valuable effective improvements of dimensional resolution are obtained. Errors in surface tension arising from various unavoidable sources such as chemical reactions or optical distortions are shown to be the main difficulties in applying the sessile drop technique at high temperature. This suggests a limiting spatial resolution of the digitizer beyond which no further improvements to the accuracy of the surface tension results are possible.

The technique has been applied to determine the surface tension of liquid Cu, Bi and Ag as well as a series of sodium borosilicate glasses.     

Calibration of Stochastic Computer Simulators Using Likelihood Emulation

We calibrate a stochastic computer simulation model of “moderate” computational expense. The simulator is an imperfect representation of reality, and we recognize this discrepancy to ensure a reliable calibration. The calibration model combines a Gaussian process emulator of the likelihood surface with importance sampling. Changing the discrepancy specification changes only the importance weights, which lets us investigate sensitivity to different discrepancy specifications at little computational cost. We present a case study of a natural history model that has been used to characterize UK bowel cancer incidence. Datasets and computer code are provided as supplementary material.     

Pretreatment Strategies for SERS Analysis of Indigo and Prussian Blue in Aged Painted Surfaces

Surface-enhanced Raman scattering (SERS) spectroscopy is increasingly applied to the identification of organic colorants in cultural heritage objects because vibrational fingerprints can be measured from microscopic samples. However, the development of SERS into a reliable, broad-spectrum method for art analysis requires the study of a wide variety of organic and inorganic colorants as well as colorant mixtures in paint. Here, we demonstrate reliable protocols for SERS-based identification of insoluble indigo, Prussian blue (PB), and mixtures thereof in aged painted surfaces. The use of simple salts and acids for sample pretreatment is evaluated. High-quality SERS spectra of PB and indigo are elucidated upon sample pretreatment with H(2)SO(4). In several cases, SERS spectra of the colorants could not be obtained without sample pretreatment. We demonstrate the use of H(2)SO(4) to solubilize PB as well as perform an in situ conversion of insoluble indigo to soluble indigo carmine (IC) on indigo, indigo oil paint, and actual samples from historic painted surfaces. A microscopic H(2)SO(4)-treated sample from the Portrait of Evelyn Byrd produced a SERS spectrum that is consistent with a mixture of PB and IC. To our knowledge, this work represents the first SERS spectrum of indigo in oil paint and the first simultaneous detection of a mixture of blue organic and inorganic colorants in a single art sample using SERS.     

Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site

Depressive illness has been reported to interfere with effortful processing, which requires conscious attention. The aim of this study was to evaluate divided attention in depressed patients, as a function of the degree of difficulty of the task performed. Tasks designed to measure unimodal and bimodal reaction times were presented to 10 patients with major depression and 10 normal control subjects. Performance was evaluated both before treatment when the patients were depressed and after treatment when they had recovered. Unlike the unimodal trials, the bimodal reaction time tasks were designed to evaluate decision-making under conditions in which attention was divided between two perceptual channels. Reaction times were measured under two different conditions in order to assess the extent of the response delay induced by divided attention, modality shifting, and decision processing. During simple response tasks, the depressed patients displayed significantly greater lengthening of reaction times when their attention was divided between two perceptual channels. This cross-modal delay effect occurred both for stimuli of the same modality and when shifting between modalities. The cross-modal delay effect was evident only for the choice tests in both the depressed and the recovered patients, but only the recovered patients were as accurate as the control subjects. These results suggest that the need for decision processing in depressed patients results in a failure to allocate the mental resources required to complete interchannel shifting, when attention is divided between two perceptual channels. These data are consistent with the hypothesis that attentional regulation is impaired in major depression.