Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classification.

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...

Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.The technique matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories and has been shown to be a promising method to replace conventional phenotypic identification for the majority of bacterial pathogens routinely found in clinical samples (1,3,8,15,17).For a subset of bacterial isolates, conventional techniques which mainly rely on commercial biochemical kit tests fail to provide a definitive identification of these infrequent isolates. In those cases, alternative techniques are required. That could be an expanded battery of biochemical tests, high-performance liquid chromatography (HPLC), or sequencing of housekeeping bacterial genes such as 16S rRNA genes and comparing the obtained sequences to those in databases (4). Since 16S rRNA gene sequencing is both time-consuming and costly, we wanted to assess the potential of MALDI-TOF MS, which is based on the study of proteic profiles, to replace it.We investigated a collection of 1,405 isolates on which 16S rRNA gene sequencing was performed in the last 8 years in our laboratory due to a lack of satisfactory identification by conventional methods such as Vitek2 or the API system (bioMérieux, Marcy l'Etoile, France). We selected for each species a maximum of 5 isolates. This yielded a total of 433 bacterial isolates representing 207 different species from 84 different genera. For 23 isolates, no growth could be obtained from frozen stock. A total of 410 isolates were consequently further studied. Acquisition of protein mass spectra of strains by MALDI-TOF MS was done on a Microflex LT instrument (Bruker Daltonics, Leipzig, Germany) using the FlexControl 3.0 software (Bruker Daltonics) after a formic acid-acetonitrile extraction step was performed as previously described (1). Automated data analysis of raw spectra was performed by the BioTyper automation 2.0 software (Bruker Daltonics), using a library of 3,290 spectra (database update of 2 September 2008) with default settings. According to the criteria proposed by the manufacturer, a result was considered valid at the species level whenever the score value attributed by BioTyper was x Ն 2.0, valid at the genus level when the score was 1.7 Յ x Ͻ 2, and no reliable identification when the score was x Ͻ 1.7. 16S rRNA gene sequencing and identification were performed by standard methods (5).For routine isolates, MALDI-TOF MS has shown overall correct identification levels often greater than 85% (1,3,8,15,17). In the present study, MALDI-TOF MS yielded a score of...

Atopic dermatitis (AD) has a well-established association with skin colonization or infection by Staphylococcus aureus, which can exacerbate the disease. However, a causal relationship between specific changes in skin colonization during the first years of life and AD development still remains unclear. In this prospective birth cohort study, we aimed to characterize the association between skin colonization and AD development in 149 white infants with or without a family history of atopy. We assessed infants clinically and collected axillary and antecubital fossa skin swabs for culture-based analysis at birth and at seven time points over the first 2 years of life. We found that at age 3 months, S. aureus was more prevalent on the skin of infants who developed AD later on. S. aureus prevalence was increased on infants' skin at the time of AD onset and also 2 months before it, when compared with age-matched, unaffected infants. Furthermore, at AD onset, infants testing positive for S. aureus were younger than uncolonized subjects. In conclusion, our results suggest that specific changes in early-life skin colonization may actively contribute to clinical AD onset in infancy.

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 ...

In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Several subtypes of Mycobacterium kansasii have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of M. kansasii strains (n ‫؍‬ 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the hsp65 gene. Subtype 1 comprised 67% of the isolates (n ‫؍‬ 128), while subtypes 2 and 3 comprised 21% (n ‫؍‬ 40) and 8% (n ‫؍‬ 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (n ‫؍‬ 8). M. kansasii subtype 1 was considered pathogenic in 81% of patients, while M. kansasii subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with M. kansasii subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping M. kansasii may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.