Enteric carriage of vancomycin-resistant Enterococcus faecium in patients tested for Clostridium difficile.

OBJECTIVE: To identify independent risk factors for enteric carriage of vancomycin-resistant Enterococcus faecium (VREF) in hospitalized patients tested for Clostridium difficile toxin. DESIGN: Retrospective case-cohort study. SETTING: Tertiary-care teaching hospital. PATIENTS: Convenience sample of 215 adult inpatients who had stool tested for C difficile between January 29 and February 25, 1996. RESULTS: 41 (19%) of 215 patients had enteric carriage of VREE Five independent risk factors for enteric VREF were identified: history of prior C difficile (odds ratio [OR], 15.21; 95% confidence interval [CI95], 3.30-70.10; P < .001), parenteral treatment with vancomycin for > or = 5 days (OR, 4.06; CI95, 1.54-10.73; P = .005), treatment with antimicrobials effective against gram-negative organisms (OR, 3.44; CI95, 1.20-9.87; P = .021), admission from another institution (OR, 2.95; CI95, 1.21-7.18; P =.017), and age > 60 years (OR 2.57; CI95, 1.13-5.82; P = .024). These risk factors for enteric VREF were independent of the patient's current C difficile status. CONCLUSIONS: Antimicrobial exposures are the most important modifiable independent risk factors for enteric carriage of VREF in hospitalized patients tested for C difficile.     

A New High-Level Gentamicin Resistance Gene,aph(2")-Id,in Enterococcus spp.

Enterococcus casseliflavus UC73 is a clinical blood isolate with high-level resistance to gentamicin. DNA preparations from UC73 failed to hybridize with intragenic probes for aac(6′)-Ie-aph(2")-Ia and aph(2")-Ic. A 4-kb fragment from UC73 was cloned and found to confer resistance to gentamicin in Escherichia coli DH5α transformants. Nucleotide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminoglycoside-modifying enzyme APH(2")-Ic and to the C-terminal domain of the bifunctional enzyme AAC(6′)-APH(2"). The gene is designated aph(2")-Id, and its observed phosphotransferase activity is designated APH(2")-Id. A PCR-generated intragenic probe hybridized to the genomic DNA from 17 of 118 enterococcal clinical isolates (108 with high-level gentamicin resistance) from five hospitals. All 17 were vancomycin-resistant Enterococcus faecium isolates, and pulsed-field typing revealed three distinct clones. The combination of ampicillin plus either amikacin or neomycin exhibited synergistic killing against E. casseliflavus UC73. Screening and interpretation of high-level aminoglycoside resistance in enterococci may need to be modified to include detection of APH(2")-Id.High-level gentamicin resistance (MIC ≥ 2,000 μg/ml) in enterococci is known to be associated with the aac(6′)-Ie-aph(2")-Ia gene, which encodes the bifunctional aminoglycoside-modifying enzyme AAC(6′)-APH(2") (14). The presence of this gene eliminates the synergism between a cell wall-active agent, such as ampicillin or vancomycin, and virtually all commercially available aminoglycosides—including gentamicin, tobramycin, netilmicin, kanamycin, and amikacin—except streptomycin (17). aph(2")-Ic is a midlevel gentamicin resistance gene (MIC = 256 μg/ml), found less commonly than aac(6′)-Ie-aph(2")-Ia in enterococci, that eliminates the synergism between ampicillin and gentamicin (6). We describe a new high-level gentamicin resistance gene initially found in Enterococcus casseliflavus that is distinct from aac(6′)-Ie-aph(2")-Ia and aph(2")-Ic.(This work was presented in part at the Infectious Diseases Society of America 34th Annual Meeting, New Orleans, La., 18 to 20 September 1996 [25], and the 97th General Meeting of the American Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997 [26].)     

Development of a standardized screening method for detection of vancomycin-resistant enterococci.

The incidence of vancomycin resistance among enterococci is increasing in the United States and elsewhere in the world, but automated susceptibility testing methods have difficulty detecting resistance expressed by certain strains. The agar screening method described by Willey et al. (B. M. Willey, B. N. Kreiswirth, A. E. Simor, G. Williams, S. R. Scriver, A. Phillips, and D. E. Low, J. Clin. Microbiol. 30:1621-1624, 1992) has been proposed as a reliable method for confirming vancomycin resistance. In this study, we investigated various parameters associated with the agar screening method and, on the basis of the findings, established optimum testing conditions for the method. First, to evaluate media and vancomycin concentrations, one laboratory used Mueller-Hinton and brain heart infusion agars supplemented with 4, 6, and 8 micrograms of vancomycin per ml to test 100 genetically characterized enterococcal strains. On the basis of the results obtained, brain heart infusion agar supplemented with 6 micrograms of vancomycin per ml was selected for further study. Subsequently, eight laboratories used the medium to test both reference and clinical isolates. There was very good performance with the reference strains and, among 158 clinical isolates tested, the method demonstrated sensitivity and specificity of 100% and from 96 to 99%, respectively.     

Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo. Received: 15 February 1997 / Accepted: 18 March 1997     

EANO guideline on the diagnosis and management of meningiomas

Meningiomas are the most common intracranial tumors. Yet, only few controlled clinical trials have been conducted to guide clinical decision making, resulting in variations of management approaches across countries and centers. However, recent advances in molecular genetics and clinical trial results help to refine the diagnostic and therapeutic approach to meningioma. Accordingly, the European Association of Neuro-Oncology (EANO) updated its recommendations for the diagnosis and treatment of meningiomas. A provisional diagnosis of meningioma is typically made by neuroimaging, mostly magnetic resonance imaging. Such provisional diagnoses may be made incidentally. Accordingly, a significant proportion of meningiomas, notably in patients that are asymptomatic or elderly or both, may be managed by a watch-and-scan strategy. A surgical intervention with tissue, commonly with the goal of gross total resection, is required for the definitive diagnosis according to the WHO classification. A role for molecular profiling including gene panel sequencing and genomic methylation profiling is emerging. A gross total surgical resection including the involved dura is often curative. Inoperable or recurrent tumors requiring treatment can be treated with radiosurgery, if the size or the vicinity of critical structures allows that, or with fractionated radiotherapy (RT). Treatment concepts combining surgery and radiosurgery or fractionated RT are increasingly used, although there remain controversies regard timing, type, and dosing of the various RT approaches. Radionuclide therapy targeting somatostatin receptors is an experimental approach, as are all approaches of systemic pharmacotherapy. The best albeit modest results with pharmacotherapy have been obtained with bevacizumab or multikinase inhibitors targeting vascular endothelial growth factor receptor, but no standard of care systemic treatment has been yet defined.     

VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405.

Enterococcus faecalis BM4405 was resistant to low levels of vancomycin (MIC, 16 microg/ml) and was susceptible to teicoplanin (MIC, 0.5 microg/ml). No PCR product was obtained when the total DNA of this clinical isolate was used as a template with primers specific for glycopeptide resistance genes vanA, vanB, vanC, and vanD. However, a 604-bp PCR fragment was obtained when V1 and V2 degenerate primers were used and total DNA was digested with HindIII as a template. The product was cloned and sequenced. The deduced amino acid sequence had greater identity (55%) with VanC than with VanA (45%), VanB (43%), or VanD (44%). This was consistent with the fact that BM4405 synthesized peptidoglycan precursors that terminated in D-serine residues. After induction with vancomycin, weak D,D-dipeptidase and penicillin-insensitive D,D-carboxypeptidase activities were detected in cytoplasmic extracts of BM4405, whereas a serine racemase activity was found in the membrane preparation. This new type of acquired glycopeptide resistance was named VanE.     

Prognostic impact of genetic alterations and methylation classes in meningioma

Meningiomas are classified based on histological features, but genetic and epigenetic features are emerging as relevant biomarkers for outcome prediction and may supplement histomorphological evaluation. We investigated meningioma‐relevant mutations and their correlation with DNA methylation clusters and patient survival times. Formalin‐fixed and paraffin‐embedded samples of 126 meningioma patients (WHO grade I 52/126; 41.3%; WHO grade II: 48/126; 38.1%; WHO grade III: 26/126; 20.6%) were investigated. We analyzed NF2, TRAF7, KLF4, ARID, SMO, AKT, TERT promotor, PIK3CA, and SUFU mutations using panel sequencing and correlated them to DNA methylation classes (MC) determined using 850k EPIC arrays. The TRAKL mutation genotype was characterized by the presence of any of the following mutations: TRAF7, AKT1, and KLF4. Survival data including progression‐free survival (PFS) and overall survival (OS) was retrieved from chart review. Mutations were evident in 90/126 (71.4%) specimens with mutations in NF2 (39/126; 31.0%), TRAF7 (39/126; 31.0%) and KLF4 (25/126; 19.8%) being the most frequent ones. Two or more mutations were observed in 35/126 (27.8%) specimens. While TRAKL was predominantly found in benign MC, NF2 was associated with malign MC (p < 0.05). TRAF7, KLF4, and TRAKL mutation genotype were associated with improved PFS and OS (p < 0.05). TERT promotor methylation, intermediate, and malign MC were associated with impaired PFS and OS (p < 0.05). Methylation cluster showed better prognostic discrimination for PFS and OS (c‐index 0.77/0.75) than each of the individual mutations (c‐index 0.63/0.68). In multivariate analysis correcting for age, gender, MC, and WHO grade, none of the individual mutations except TERT remained an independent significant prognostic factor for PFS. Molecular profiling including mutational analysis and DNA methylation classification may facilitate more precise prognostic assessment and identification of potential targets for personalized therapy in meningioma patients.     

Activities of carbapenem and comparator agents against contemporary US Pseudomonas aeruginosa isolates from the CAPITAL surveillance program

Antimicrobial susceptibilities of contemporary Pseudomonas aeruginosa clinical isolates were determined from the CAPITAL 2010 surveillance program. Isolates were collected from 100 sites throughout the USA and Puerto Rico, and included isolates representing a range of patient demographics and infection types. A total of 2722 isolates were tested for susceptibility to a broad spectrum of agents, with susceptibilities ranging from 98.8% for colistin to 74% for levofloxacin. Doripenem was the most active carbapenem agent, with 88.6% of isolates susceptible, in comparison with 78.1% and 84.6% for imipenem and meropenem, respectively. Lower respiratory tract isolates and isolates from the intensive care unit setting were the least susceptible overall. Resistance rates were typically highest in lower respiratory tract isolates, with the exception of urinary tract isolates, which displayed the highest resistance for levofloxacin. Overall, multidrug-resistant isolates comprised 14.8% of the total sample population.     

Light and electron microscopic examination of endothelial cells from bovine retinal vessels in long-term cultures

The aim of the present work was to examine and compare the ultrastructure of bovine retinal endothelial cells (BRECs) in vitro during several passages in a medium selective for endothelial cells. The identity of the endothelial cells was confirmed immunohistochemically, up to the tenth passage. Changes in their ultrastructure in comparison to endothelial cells in vivo occurred at the onset of culturing and not progressively with repeated passages. The cultured BRECs show high metabolic activity in all passages. While retaining their identity as endothelial cells, they modify their lipid metabolism, so that lipids are stored. This change in lipid metabolism was induced by the medium. Received: 19 May 1999 / Accepted: 4 August 1999