Use of Maldi-Tof Mass spectrometry in direct microorganism identification in clinical laboratories

Abstract

protecting them from the disruptive energy of the laser. The type of mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer). TOF is a revelation system that relates the time of flight of a ion to the charge/mass value: ion with a higher m/z have are slower (a bigger time of flight) than ions with lower m/z. MALDI-TOF instruments are usually equipped with an ion mirror, deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution. After separation, ions go to the detector and the data are collected in an electronic sheet. The procedure generates protein fingerprint signatures from whole bacterial cells called "peptide spectra"; a "unique spectrum" corresponds to a microorganism ("fingerprint spectrum"). The identification of microorganisms is obtained by comparing, using algorithms, the obtained spectrum to a database containing spectra that are generated based on measurements on well known microoorganisms. These spectra contain characteristic signals of genus, species and strain biomarkers which are representative for the respective group of microorganisms. At present MALDI-TO MS allows the identification of most bacteria and an increasing number of fungi. For routine bacterial isolates, a correct identification by MALDI-TOF MS at the species level was obtained in 79.9% to 93.6% of cases (2). Commercially available MALDI-TOF MS devices designed for clinical microbiology laboratories are of a size of classical laboratory device and do not require specific equipment other than a computer. They can also connect to the laboratory information system or to the other automated devices (such as the spotter that automatically spots the bacteria containing solution on the sample tray). Two MALDI-TOF MS system are commercially available: one is from Bruker Daltonics and the other is from Shimadzu Corporation. The comparison between the two instruments has shown little differences in microorganism identification (from colonies); in fact the Bruker system gave high confidence identification for 680 isolates of 720 (of which 674, 99.1%, were correct) and the Shimadzu system gave high confidence identification for 639 isolates (of which 635, 99.4%, were correct) (4). DIRECT IDENTIFICATION OF MICROORGANISMS The identification of microorganisms can be performed directly from body fluids (e.g. urine, blood culture, after centrifugation and recovery of microorganisms) or from colonies (after cultivation) (3). Data from literature on body fluids are mainly obtained in blood cultures, but also the identification directly from urine is of interest. As far as blood cultures are concerned, a number of papers has been published (7, 10, 11, 15). This interest is because the turnaroundtime for this "exam" is crucial: in fact the sooner the appropriate therapy begins the better are the outcomes. Pathogen growth in blood culture vessels is continously monitored by semiautomated incubators. At present, on cultures recognized as positive, Gram's stain allows for preliminary identification. Positive cultures with one microorganism are processed in a different way than those with more than one microorganism. In positive monomicrobial cultures, after separation of microbs from blood cells, we can perform an antiobiogram susceptibility test. This procedure permits to obtain an immediate identification that we can communicate to the clinician, and gives indication to perform the correct antibiogram. Major problems are present when more than one microorganism are in the culture: in this case we have to use the method of subcultivation and then the identification with mass-spectrometry can be performed. Also in this case, this method is less time-consuming than the classical one, because as soon as the colonies are grown we can have identification without waiting for the time that we need to perform it with the classical method (that means that 24 post-positivisation of a culture with more than one microb (Figure II). The time between positivisation and the final result (identification + antibiogram) is, in both cases, significantly reduced: with MALDI-TOF it arrives in 24/48 hours, without MALDI-TOF we need 48/72 hours. This approach using MALDI is very useful in terms of reducing the time to have a result and consequently in terms of reducing the time between positivisation of blood culture and beginning of the appropriate therapy. When fungi are present in blood cultures direct MALDI-OF MS analysis of aliquots from positive cultures allows rapid and accurate identification of the main Candida species, thus