Tuesday, March 15, 2011

MALDI-TOF Mass Spectrometry and Equivalency Testing

Rosa Siciliano, CNR, provided an overview of MALDI-TOF mass spectrometry. Microbial identification is based on spectral fingerprints between different microorganisms, where some peaks (molecular masses) are specific to Genus, species, and in some cases, subspecies characterization. Additionally, spectra are reproducible as long as the bacteria are grown under the same conditions. Mass spectra are derived from ribosomal proteins, although spectra of surface macromolecules can also be obtained. The strengths of using MALDI-TOF mass spectrometry include the identification of bacterial, spores, yeast and mold, automated analysis, and no need for preliminary information prior to evaluation (e.g., Gram stain result). Some weaknesses include the potential need to grow bacteria to levels that can be detected by the system, operators are required to have a basic level of expertise in mass spectrometry, and it is not possible to obtain multiple identifications from mixed cultures (i.e., a pure culture is currently required).

Mark Blanchard and Deborah Smith, both from Merck Millipore, provided insights on how to demonstrate equivalence between an existing method and an alternative method. They presented three examples of how to demonstrate equivalence: (a) accuracy of matched outcomes, (b) comparing two independent assays near the limit of detection, and (c) evaluating the false negative rate of a presence/absence test. The speakers also discussed the limitations when using low levels of microorganisms (e.g., less than 5 cfu) when demonstrating equivalence between two methods: many of the counts may actual be negative (i.e., 0 cfu), the percent variation is high, and you will need many replicates to accurately estimate the average cell count. The discussions will form the basis of a workshop on this topic tomorrow afternoon. Stay tuned for additional entries!


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