Evaluation of imipenem/imipenem+EDTA disk method for detection of metallo-beta-lactamase-producing Klebsiella pneumoniae isolated from blood cultures.

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla (VIM-1) gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/ml were found positive for the bla (VIM-1) gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 microg/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.

An improved flow cytometric assay for detection and discrimination between malignant cells and atypical mesothelial cells, in serous cavity effusions.

BACKGROUND: The aim of this study was to evaluate a flow cytometric assay for the detection of malignant effusions. METHODS: During the last 4-year period, 125 effusions suspicious for malignancy were prospectively analyzed by flow cytometry and conventional cytology. A three-step flow cytometric assay was performed, beginning with an initial informative panel of two protocols, containing SYTO-16, 7-AAD, CD71-PE, CD45-ECD, and CD66abce-FITC, CD64-PE, CD45-ECD, CD16-PECy5, CD14-PECy7, respectively. This was followed by a basic immunophenotypic panel of seven three-color combinations, containing in the first position, EMA, Ber-EP4, CD66abce, CD56, and intracellular desmin-33, combined with CD71-PE and CD45-PeCy5 in each tube. Finally, a cytokeratin-FITC/propidium iodide DNA panel was conducted, for the detection of aneuploidy in cytokeratin positive cells. RESULTS: The sensitivity and specificity of flow cytometry were 85.1 and 97.8%, and of cytology 93.2 and 95.6%, respectively. A significant association was observed between the results of the two techniques (P < 0.001). Among eight atypical cases detected by cytology, five had been precisely characterized as malignant by flow cytometry. EMA and Ber-EP4 proved the most sensitive markers for malignancy diagnosis, while the detection of desmin-33 negative/cytokeratin positive cells had the simultaneous highest positive and negative predictive values. CD66abce was very specific, although nonsensitive, while DNA ploidy analysis was nonspecific, as hyperploidy was observed in reactive mesothelial cells. CONCLUSIONS: A flow cytometric assay of high sensitivity and specificity is proposed for the routine identification of carcinoma cells in effusions and their distinction from atypical mesothelial cells, as an ancillary to conventional cytology.

Detection of metallo-ß-lactamase genes in clinical specimens by a commercial multiplex PCR system.

hyplex®-MBL ID Multiplex PCR-ELISA, a novel method for identifying metallo-ß-lactamase genes directly in clinical specimens, was evaluated using a consecutive collection of 326 samples from three hospitals in Greece characterized by high prevalence of VIM producers. The method exhibited high sensitivity (98.0%) and specificity (98.6%) and was proven reliable in detecting bla(VIM) genes in blood, urine, pus, and sputum samples that, as confirmed by conventional methods, contained various VIM-producing species. Future multicenter studies should be considered for the thorough evaluation of this method and its potential diagnostic utility.

Multiple liver abscesses associated with bacteremia due to Leuconostoc lactis.

Leuconostoc species, which are members of the family Streptococcacae, have only recently been recognized as potential pathogens. We describe a patient with type II diabetes mellitus who had multiple liver abscesses associated with bacteremia due to Leuconostoc lactis. To our knowledge, this is the first case of this association to be reported in the literature.