Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 ؋ g) to remove leukocytes and then at high revolutions (15,500 ؋ g) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >10 5 CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was Escherichia coli (173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%).Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved.Urinary tract infections (UTIs) are among the most common human bacterial infections. More than 80% of uncomplicated UTIs are caused by Escherichia coli (9). Other common uropathogens include Staphylococcus saprophyticus in uncomplicated UTIs and Gram-negative rods (enterobacteria other than E. coli or Pseudomonas aeruginosa) and Gram-positive cocci in complicated UTIs (9). UTIs affect an estimated 1 out of 3 women before the age of 24 (7). Up to 40 to 50% of the female population will develop a symptomatic UTI at some time during their life, and about 33% of women with acute uncomplicated UTIs have frequent recurrences (7).Several tests are available for screening patients for UTIs, including urine dipstick testing, urinalysis, Gram staining, and urine culture. Urine culture is the "gold standard" for defining the diagnosis of UTIs, because it allows the quantification and identification of the uropathogenic species. However, this method is time-consuming and expensive. Up to 70% of urine cultures are negative, with high costs for unnecessary testing (4). Thus, automated analyzers for UTI screening to rapidly identify negative samples that can be reported promptly to clinicians are now widely used. Samples positive in t...

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows a fast and reliable bacterial identification from culture plates. Direct analysis of clinical samples may increase its usefulness in samples in which a fast identification of microorganisms can guide empirical treatment, such as blood cultures (BC). Three hundred and thirty BC, reported as positive by the automated BC incubation device, were processed by conventional methods for BC processing, and by a fast method based on direct MALDI-TOF MS. Three hundred and eighteen of them yield growth on culture plates, and 12 were false positive. The MALDI-TOF MS-based method reported that no peaks were found, or the absence of a reliable identification profile, in all these false positive BC. No mixed cultures were found. Among these 318 BC, we isolated 61 Gram-negatives (GN), 239 Gram-positives (GP) and 18 fungi. Microorganism identifications in GN were coincident with conventional identification, at the species level, in 83.3% of BC and, at the genus level, in 96.6%. In GP, identifications were coincident with conventional identification in 31.8% of BC at the species level, and in 64.8% at the genus level. Fungaemia was not reliably detected by MALDI-TOF. In 18 BC positive for Candida species (eight C. albicans, nine C. parapsilosis and one C. tropicalis), no microorganisms were identified at the species level, and only one (5.6%) was detected at the genus level. The results of the present study show that this fast, MALDI-TOF MS-based method allows bacterial identification directly from presumptively positive BC in a short time (<30 min), with a high accuracy, especially when GN bacteria are involved.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory.

The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses, is believed to occur by direct fusion at the plasma membrane through a pH-independent mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when drugs were added after virus adsorption. Cells treated with methyl b-cyclodextrin, a drug that sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus-cell binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies against the early endosome marker EEA1 revealed the localization of the virus in these intracellular structures. Using fluorescence microscopy, it was found that cell-cell fusion was enhanced at low pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway, suggesting that this pathway could be an alternative route for virus entry into cells. INTRODUCTIONNewcastle disease virus (NDV) is one of the causes of avian respiratory diseases and economic losses in the poultry industry. It is a member of the family Paramyxoviridae, which includes enveloped, negative-stranded RNA viruses that encode two transmembrane glycoproteins: an attachment protein, HN, and a fusion protein or F protein. Virus adsorption occurs at the surface of the host cell membrane. The viral attachment protein HN recognizes and binds to sialic acid-containing molecules such as glycoproteins and glycolipids (Ferreira et al., 2004) and has neuraminidase and fusion-promoting activities. Following binding, the F protein promotes fusion between the viral and cellular membranes and the viral genome is delivered into the cytoplasm.Enveloped viruses enter the cell through two main pathways: direct fusion between the viral envelope and the plasma membrane; and receptor-mediated endocytosis. In the case of paramyxoviruses, it has been established that the membrane fusion process takes place at the host plasma membrane in a pH-independent manner. Despite this, it has been shown previously that fusion of NDV with cultured cells is enhanced at acidic pH (San Roman et al., 1999), leading us to propose that NDV may also penetrate the cell via an endocytic pathway in a pH-dependent process.After binding of an enveloped virus to its cognate receptor at the cell surface, membrane fusion is responsible for delivery of the nucleocapsid into the cytoplasm. Receptor binding and low pH have been considered to be the two main triggering mechanisms responsible for conformational changes in viral envelope glycoproteins leading to fusion. Also, it has been shown that in the recently emerged paramyxoviruses Nipah virus and Hendra virus, activation of the F protein is accomplished by proteolysis after endocytic entr...

BackgroundMALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures.Methodology/Principal FindingsWe created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct.Conclusions/SignificanceMALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.