Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification. MALDI-TOF mass spectrometry yielded 96.3% and 84.5% accurate species level identifications (spectral scores, >1.8) for 138 common and 103 archived strains of yeast. MALDI-TOF mass spectrometry is accurate, rapid (5.1 min of hands-on time/identification), and cost-effective ($0.50/sample) for yeast identification in the clinical laboratory.Candida is the fourth leading cause of nosocomial bloodstream infections in the United States (22). Candida albicans is the major species causing morbidity and mortality, but other, less common opportunistic yeasts, including non-albicans Candida, Cryptococcus, Pichia, Rhodotorula, Trichosporon, and Saccharomyces species have also been seen in immunocompromised settings (4,15,31). Accurate and rapid identification of yeasts is critical for treatment due to species-specific susceptibility patterns. Phenotypic identification can be timeand labor-intensive and can at times yield erroneous identifications (6,20,24,30). Rapid latex agglutination assays are available, but for only a limited number of species (12, 23). Molecular assays are often accurate but can be expensive and complex.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been described as a rapid, reliable, and cost-effective alternative for bacterial, mycobacterial, and fungal identification (1,10,19,21,27,29). The technique relies on the generation of microorganism "protein fingerprints" that are compared to reference spectra in a well-characterized library (9).In this study, we assessed the performance of the MALDI Biotyper 2.0 Microflex LT spectrometer (Bruker Daltonics, Inc., Billerica, MA) for the identification of common and uncommon yeasts (n ϭ 261). A 1-month, blinded, prospective study included 145 freshly collected yeast isolates encountered in the routine laboratory workflow on various selective fungal isolation media, including Mycosel agar, brain heart infusion (BHI), Sabouraud's (SAB) agar, and inhibitory mold agar (IMA) (Becton Dickinson, Sparks, MD) (17). C. albicans was identified by germ tube formation, and Candida glabrata was identified by the rapid-assimilation-of-trehalose (RAT) test (17). Both tests have inherent limitations, including false-positive germ tube formation by non-albicans species (e.g., Candida dubliniensis) and the need to carefully control the inoculum size (11, 16). Round, variably-sized yeasts resemblingCryptococcus were identified at the species level using urea and pigment production methods, as described previously (14). All other yeasts were identified using the API 20C AUX yeast identification system (bioMérieux, Hazelwood, MO) and microscopic morphology on cornmeal agar with Tween 80 (17). Additionally, 116 strains (IMA subcultures at 30°C) from an archived collection of less common yeasts that were previously identified using API 20C and D2 sequencing were identified usi...

An evaluation of the MicroSeq 500 microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Evaluation of the MicroSeq 500 microbial identification system was accomplished with 59 American Type Culture Collection (ATCC) strains and 328 clinical isolates of mycobacteria identified by conventional and 16S ribosomal DNA sequencing by using the MicroSeq 500 microbial identification system. Nucleic acid sequencing identified 58 of 59 (98.3%) ATCC strains to the species level or to the correct group or complex level. The identification results for 219 of 243 clinical isolates (90.1%) with a distance score of <1% were concordant with the identifications made by phenotypic methods. The remaining 85 isolates had distance scores of >1%; 35 (41.1%) were identified to the appropriate species level or group or complex level; 13 (15.3%) were identified to the species level. All 85 isolates were determined to be mycobacterial species, either novel species or species that exhibited significant genotypic divergence from an organism in the database with the closest match. Integration of nucleic acid sequencing into the routine mycobacteriology laboratory and use of the MicroSeq 500 microbial identification system and Mayo Clinic databases containing additional genotypes of common species and added species significantly reduced the number of organisms that could not be identified by phenotypic methods. The turnaround time was shortened to 24 h, and results were reported much earlier. A limited number of species could not be differentiated from one another by 16S ribosomal DNA sequencing; however, the method provides for the identification of unusual species and more accurate identifications and offers the promise of being the most accurate method available.During the past two decades, clinical mycobacteriology has enjoyed several important advances that have improved the means for the detection and identification of mycobacteria from clinical specimens. Ultimately, all have improved patient care by shortening turnaround times in the laboratory, and the advances continue. For many years traditional phenotypic identification methods have been used to identify mycobacteria. All are slow, many provide equivocal results, and identifications are not always timely and accurate. High-performance liquid chromatography (HPLC) was used by state laboratories and some reference laboratories. This provided for the rapid identification of an expanded list of species of mycobacteria. Gas-liquid chromatography was also developed and proved to be a useful identification method; however, the number of species that could be identified by gas-liquid chromatography was less compared to the number that could be identified by HPLC. The development of nucleic acid probes for confirmation of the identity of the isolate had a major impact on the work flow in many laboratories. Probes were developed for the identification of members of the Mycobacte...

The value of matrix-assisted laser desorption ionization؊time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed. C urrently, there are more than 170 recognized species and subspecies of mycobacteria, more than 100 Nocardia species, and several hundred other aerobic actinomycetes species distributed across approximately 16 genera (1). Some of these organisms are clinically relevant and cause a spectrum of disease presentations in humans, while others are environmental organisms that can be found as commensal organisms or in laboratory cultures as contaminants resulting from specimen collection or processing (2). Accurate identification of mycobacteria and the aerobic actinomycetes is important for patient care but can be difficult due to the low growth rates of some species, the large number of species which have small differences in genetic diversity, and the need for biosafety level (BSL) 3 facilities when unknown isolates that might be Mycobacterium tuberculosis or Mycobacterium bovis are processed. The current gold standard for the identification of mycobacteria and aerobic actinomycetes is DNA sequencing with several targets recognized as useful for the species identification of mycobacteria and aerobic actinomycetes, including the 16S rRNA gene, rpoB, secA, and hsp65 (3). However, many clinical laboratories do not have the resources to routinely perform sequencing because it is labor-intensive and technically complex.In the last few years, matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) has proven to be a reliable method for the identification of a wide variety of bacteria and yeasts following growth on culture medium (4-8). Fewer studies have ...

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Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi. Cryptococcus neoformans is ecologically widespread and affects primarily immunocompromised patients, while C. gattii is traditionally found in tropical climates and has been reported to cause disease in immunocompetent patients. L-Canavanine glycine bromothymol blue (CGB) agar can be used to differentiate C. neoformans and C. gattii, but there are few reports of its performance in routine clinical practice. Growth of C. gattii on CGB agar produces a blue color, indicating the assimilation of glycine, while C. neoformans fails to cause a color change. Using reference and clinical strains, we evaluated the ability of CGB agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) to differentiate C. neoformans and C. gattii. One hundred two yeast isolates were screened for urease activity, melanin production, and glycine assimilation on CGB agar as well as by D2 sequencing. Seventeen of 17 (100%) C. gattii isolates were CGB positive, and 54 of 54 C. neoformans isolates were CGB negative. Several yeast isolates other than the C. gattii isolates were CGB agar positive, indicating that CGB agar cannot be used alone for identification of C. gattii. D2 correctly identified and differentiated all C. gattii and C. neoformans isolates. This study demonstrates that the use of CGB agar, in conjunction with urea hydrolysis and Niger seed agar, or D2 LSU sequencing can be reliably used in the clinical laboratory to distinguish C. gattii from C. neoformans. We describe how CGB agar and D2 sequencing have been incorporated into the yeast identification algorithm in our laboratory.Cryptococcus gattii (formerly Cryptococcus neoformans var. gattii) differs from the closely related C. neoformans in several ways, including a contrasting host profile and a reduced susceptibility to certain antifungal agents (19,20). Cryptococcus gattii has traditionally been thought to be geographically restricted to tropical and subtropical climates, although recent reports indicate that it can be found worldwide, including in regions with distinctly nontropical climates, like the Pacific Northwest (2,4,8,12,14,17,19). Clinically, the majority of cryptococcosis cases that occur in AIDS patients and other immunocompromised hosts are caused by C. neoformans var. neoformans or C. neoformans var. grubii. In contrast, C. gattii has been reported to cause meningoencephalitis and pulmonary infections in hosts who are generally immune competent (9,14,18).Since C. gattii is recognized as an emerging pathogen, it is important for the clinical microbiology laboratory to accurately differentiate it from C. neoformans. A recent proficiency testing survey administered by the New York State Department of Health indicated that only 7/140 (5%) clinical laboratories participating in the event were able to correctly identify C. gattii, while the remaining 95% of laboratories surveyed misidentified the isolate as C. neoformans (15). The critique that followed this proficiency event in...