Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.In the clinical diagnostic microbiology laboratory, the identification of bacterial or yeast isolates is currently mainly based on phenotypic characteristics, such as growth on different media, colony morphology, Gram stain, and various biochemical reactions. Altogether, these techniques allow the identification of most bacterial isolates with great accuracy, but they are costly and time-consuming.The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique can be used to generate protein fingerprint signatures from whole bacterial cells (9). By comparing these fingerprints to a database of reference spectra by the use of various algorithms, bacteria can be rapidly identified (9). Even though the first study of the use of MS for the identification of bacteria dates back to 1975 and was performed by Anhalt and Fenselau (1), MALDI-TOF MS devices designed for use under routine conditions have only recently been commercially introduced.Over the past few years, this technique has been used in specific studies that have essentially assessed its ability to identify different bacterial genera among Gram-negative rods, such as Escherichia coli and other members of the Enterobacteriaceae family (5, 6); Gram-positive cocci, such as Staphylococcus aureus and streptococci (8, 12); and some Gram-positive rods, such as Bacillus cereus and Listeria species (3, 18). Very recently, the first study to have assessed the performance of MALDI-TOF MS for the identificatio...

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.Blood cultures are the best approach to establishing the etiology of bloodstream infections and infectious endocarditis. Moreover, rapid identification of the etiological agents of such severe infections is pivotal to guiding antimicrobial therapy. Thus, the impact of timely microbiology laboratory reporting is maximal at the notification of positive blood cultures (5). Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) allows identification of both gram-positive (2, 7) and gram-negative bacteria (1, 4) to the species level in a few minutes by measuring the molecular masses of proteins and other bacterial components obtained from whole bacterial extracts. A relatively low crude bacterial load of about 5 ϫ 10 3 CFU is necessary for reliable MALDI-TOF analysis (3), suggesting that bacterial identification might be done directly with blood culture pellets. We applied a simple procedure for lysing erythrocytes from positive blood cultures and prepared a bacterial pellet for MALDI-TOF MS analysis.Pellets from positive blood culture vials (Plus aerobic/F, Lytic anaerobic/F, and Peds/F; Becton Dickinson) detected by the Bactec 9240 automated blood culture system (Becton Dickinson) were prepared as follows shortly after the automated system flagged a positive vial. Five milliliters of positive medium was added to 40 ml of sterile H 2 O. The sample was mixed and centrifuged at 1,000 ϫ g for 10 min. H 2 O and blood cells were removed (Fig. 1A). The pellet was then suspended in 1 ml of a home-made ammonium chloride lysing solution (0.15 M NH 4 Cl, 1 mM KHCO 3 ) and centrifuged at 140 ϫ g for 10 min (Fig. 1B and C). When the pellet remained hemorrhagic, the supernatant was discarded and the pellet was washed again with 2 ml of H 2 O. MALDI-TOF MS analysis was then directly performed on the bacterial pellet or after an additional extraction step. To extract proteins, 20 l of the pellet was mixed with 1 ml of 70% ethanol. After a further centrifugation at 13,000 ϫ g for 2 min, the pellet was mixed with 25 l of 70% formic acid and 25 l of pure acetonitrile. After centrifugation at 13,000 ϫ g for 2 min, 1 l of the supernatant containing the bacterial extract was transferred onto the MALDI target plate and dried. Subsequently, both unextracted and extracted samples were overlaid with 1 l of MALDI matrix (a saturated solution of ␣-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% trifluoroacetic acid) and dried in air.Mass spectra were then acquired by Microflex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). MALDI BioTyper 2.0 software was used ...

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual "prominent growth reduction" (MIC 2 ) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ؎ one dilution range across all 17 laboratories. MIC 2 at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.

Twenty-five patients seen consecutively at an HIV outpatient clinic who had clinical evidence of oropharyngeal candidiasis and two or more oral swabs positive for yeasts on culture were studied retrospectively. For each of the 65 isolates susceptibility to fluconazole was evaluated by the disk diffusion test and determination of the minimal inhibitory concentration (MIC). A correlation was sought between clinical resistance and in vitro susceptibility data. Seven patients were non-responders and 19 were responders (one patient figuring in both groups). Significant differences were observed between the two groups with respect to the median interval after the diagnosis of AIDS (27 months in non-responders and 2 months in responders; p = 0.001), the median CD4+ cell count (6 and 21 cells/mm3 respectively; p = 0.005) and the median number of previous episodes of oropharyngeal candidiasis treated with fluconazole (13 and 2 episodes respectively; p = 0.001). Candida albicans was identified in 64 of 65 cultures. The correlation between MIC values and diameters of inhibition was good (r = 0.85; p < 0.001). The degree of in vitro susceptibility of the isolates to fluconazole showed a significant difference between non-responders and responders (mean inhibition diameters 13 and 36 mm respectively; p < 0.001) with a tentative cut-off value of 25 mm. An advanced stage of HIV infection and previous exposure to fluconazole could be risk factors for the development of fluconazole-resistant oropharyngeal candidiasis. Candida albicans strains with decreased in vitro susceptibility to fluconazole were responsible for the clinical resistance which could be predicted by a simple disk diffusion test.

Twenty-one chlamydospore-forming and germ tube-positive Candida albicans clinical isolates from 15 human immunodeficiency virus (HIV)-positive and 3 HIV-negative patients were examined by two different genetic methods. Multilocus enzyme electrophoresis and hybridization with the C. albicans-specific Ca3 probe showed that such isolates can be split into two genetically distinct groups that can be clearly distinguished. One group mainly contained strains with atypical sugar assimilation patterns and could be distinguished from the other group by the absence of intracellular ␤-glucosidase activity. All 13 strains belonging to this group were isolated from the oral cavities of asymptomatic HIV-positive drug users and may be less pathogenic than the eight strains from the other group isolated either from HIV-positive patients with oropharyngeal candidiasis or from HIV-negative patients with invasive candidiasis.