An on-plate testing method using formic acid was evaluated on the Bruker Biotyper matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry system using 90 yeast and 78 Corynebacterium species isolates, and 95.6 and 81.1% of yeast and 96.1 and 92.3% of Corynebacterium isolates were correctly identified to the genus and species levels, respectively. The on-plate method using formic acid yielded identification percentages similar to those for the conventional but more laborious tube-based extraction. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a rapid and accurate method for identification of bacteria and fungi (2,3,5,6). This technology employs an ionizing laser to generate isolate-derived spectra, which are compared in real time to a library of reference spectra. Depending on the similarity between the acquired spectra and the library, an identification and a confidence score are generated. Some organisms, such as Gram-negative bacteria, are easily analyzed by directly smearing a colony onto the MS plate. Other types of bacteria and yeasts generally require preparatory tube extraction (2). Using the Bruker Biotyper MALDI-TOF MS system (Billerica, MA) and a preparatory tube extraction method, we have previously shown that most yeast and Corynebacterium species are correctly identified to the genus and species levels (1, 3). The tube extraction process involves immersing the isolate in 70% ethanol in a separate tube and pelleting and drying the organism followed by extraction and spotting onto the MS plate. This is cumbersome and time-consuming for routine clinical use, and it requires additional labeling steps, which may lead to specimen misidentification, compromising patient safety.To overcome these preanalytical processing limitations, we evaluated a rapid, direct on-plate testing method using formic acid for the identification of yeast and Corynebacterium species, and we compared our results to those from our prior studies of the same isolates tested by the preparatory tube extraction method (1, 3). Furthermore, the on-plate extraction method uses smaller volumes of formic acid and fewer laboratory consumables than tube extraction, and it is an overall more environmentally friendly process for isolate preparation.(This material was presented, in part, at the 112th American Society for Microbiology General Meeting, San Francisco, CA, 16 to 19 June 2012.)A total of 90 yeast and 78 Corynebacterium species isolates were cultured as described previously (1, 3) and analyzed using the Bruker Microflex LT/SH Biotyper following direct on-plate extraction. For yeast isolates, on-plate testing was performed by smearing a small amount of the organism from a single colony directly onto a spot on the MALDI-TOF MS steel anchor plate (BigAnchor 96-well plate; Bruker) and overlaying it with 1 l of 70% formic acid (Fluka [SigmaAldrich], St. Louis, MO). The order of addition was reversed (formic acid followed by smearing with organism) for the Co...
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 ...
ObjectivesRapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation.MethodsThe NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method).ResultsA wide range in performance (33–77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance.ConclusionSuccessful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.
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