0 évaluation0% ont trouvé ce document utile (0 vote)
16 vues9 pages
Despite widespread application of nucleic acid diagnostics, cultures remain integral in modern laboratories. Their downside is slow turn-around time, impacted by time to growth and identification of that growth. A new proteomic technology, MALDI-TOF mass spectrometry, expedites this process.
Despite widespread application of nucleic acid diagnostics, cultures remain integral in modern laboratories. Their downside is slow turn-around time, impacted by time to growth and identification of that growth. A new proteomic technology, MALDI-TOF mass spectrometry, expedites this process.
Despite widespread application of nucleic acid diagnostics, cultures remain integral in modern laboratories. Their downside is slow turn-around time, impacted by time to growth and identification of that growth. A new proteomic technology, MALDI-TOF mass spectrometry, expedites this process.
M E D I C A L M I C R O B I O L O G Y I N V I T E D A R T I C L E
Melvin P. Weinstein and L. Barth Reller, Section Editors
Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry in Clinical Microbiology Robin Patel Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology and Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, Minnesota Despite widespread application of nucleic acid diagnostics, cultures remain integral in modern laboratories. Because cultures detect a large number of organism types, it is unlikely that they will disappear from clinical practice in the near future. Their downside is slow turn-around time, impacted by time to growth and identi- cation of that growth. The latter is expedited using a new proteomic technology, matrix-assisted laser desorp- tion ionizationtime of ight mass spectrometry (MALDI-TOF MS). Keywords. mass spectrometry; bacteria; fungi; MALDI-TOF; matrix-assisted laser desorption ionization-time of ight. Despite widespread application of nucleic acid diagnos- tics, cultures remain integral in modern laboratories. Because cultures detect a large number of organism types, it is unlikely that they will disappear from clinical practice in the near future. Their downside is slow turn-around time, impacted by time to growth and identication of that growth. The latter is expedited using a new proteomic technology, matrix-assisted laser desorption ionizationtime of ight mass spec- trometry (MALDI-TOF MS). Historically, identication of cultured bacteria and fungi has been a complex, algorithmic task. Textbooks, owcharts, and tables have been used to interpret colony morphology as well as stains and biochemical tests performed on colonies. Phenotypic tests may be contingent on subsequent growth, prolonging time to identication. Manual biochemical tests (eg, coagulase, catalase) are rapid, but identify limited organism types (eg, Staphylococcus aureus). Biochemical test panels performed manually (eg, API strips, bioMrieux, Dur- ham, North Carolina) or on automated instruments (eg, VITEK, bioMrieux; BD Phoenix Automated Mi- crobiology System, BD, Franklin Lakes, New Jersey; Mi- croScan, Siemens Healthcare Diagnostics, Tarrytown, New York) identify more microorganisms, but have longer turn-around times and costly consumables, and often require knowledge of the organism type being tested (eg, gram-positive cocci). Finally, broad-range PCR followed by sequencing accurately identies bacte- ria and fungi but has a long turn-around time, and is expensive and performed in select laboratories. With MALDI-TOF MS, colonies growing in culture are inex- pensively and accurately identied in minutes, without a priori knowledge of microorganism type; users do not even need to know whether a bacterium or a yeast is being tested. The product of advances in mass spec- trometry and bioinformatics, MALDI-TOF MS is revo- lutionizing bacterial and fungal identication in clinical microbiology [1]. The concept of using mass spectrometry to identify bacteria (albeit not based on proteomics) was proposed in the 1970s [2]; with the Nobel Prizewinning invention of MALDI-TOF MS in the 1980s [3, 4], whole-organism Received 29 January 2013; accepted 5 April 2013; electronically published 17 April 2013. Correspondence: Robin Patel, MD, Mayo Clinic, Division of Clinical Microbiology, 200 First St SW, Rochester, MN 55905 ( patel.robin@mayo.edu). Clinical Infectious Diseases 2013;57(4):56472 The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. DOI: 10.1093/cid/cit247 564 CID 2013:57 (15 August) MEDICAL MICROBIOLOGY
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
proteomics became possible. It took until the 1990s for develop- ment of libraries of reference spectra and software for bacterial identication, and until the past few years for commercialization of the technology [1, 5]. MALDI-TOF MS, used for a few years in European clinical microbiology laboratories, and being rap- idly adopted worldwide, is likely to antiquate most biochemical microbial identication in the near future. The simplest MALDI-TOF MS applications test bacterial or yeast colonies without elaborate preparation and using minimal consumables, providing rapid and low-cost identication. Testing begins by picking an unknown colony from a culture plate to a spot on a MALDI-TOF MS target plate (Figure 1). The cells may be treated on the target plate (we use formic acid) and are ultimately overlain with 12 L of matrix. Follow- ing a short drying period, the plate is placed in the ionization chamber of the mass spectrometer (Figure 2). MALDI refers to the matrix, which assists in the desorp- tion and ionization of microbial analytes through pulses of energy from an ultraviolet N 2 laser. The matrix (eg, -cyano-4- hydroxycinnamic acid dissolved in 50% acetonitrile and 2.5% triuoroacetic acid) isolates microbial molecules from each other; microbial and matrix molecules on the target plate are desorbed, with the majority of energy being absorbed by the matrix, converting it to an ionized state. Through random colli- sion in the gas phase, charge is transferred from matrix to mi- crobial molecules; analytes within the mass range specied are measured, the most abundant being ribosomal proteins. The cloud of ionized microbial proteins is funneled through a posi- tively charged electrostatic eld into a time of ight or TOF mass analyzer, a tube under vacuum. The ions travel toward an ion detector with small analytes traveling fastest, followed by progressively larger analytes. A mass spectrum is generated, representing the number of ions of a given mass impacting the ion detector over time. Analytes are not individually characterized; instead, the gen- erated mass spectrum provides a prole (ngerprint) of the unknown microorganism. Generated mass spectra are unique to individual microorganism types, with peaks specic to genera, species, and even strains. The test isolates mass spec- trum is automatically compared to a database of reference Figure 1. Matrix-assisted laser desorption ionizationtime of ight mass spectrometry (MALDI-TOF MS) workow. A bacterial or fungal colony (typically single) is picked from a culture plate to a spot on a MALDI-TOF MS target plate (a reusable or disposable plate with a number of test spots) using a wooden or plastic stick, pipette tip, or loop. One or many isolates may be tested at one time. At our institution, cells are treated with formic acid on the target plate. The spot is then overlain with 12 L of matrix. Following a short drying period, the plate is placed in the ionization chamber of the mass spectrometer for analysis (see Figure 2). A mass spectrum is generated and automatically compared against a database of mass spectra by the software, resulting in identi- cation of the organism (Candida parapsilosis in position A4 in the example). More complex preparatory protein extraction, commonly used in older studies, may be performed prior to target plate inoculation. Although this yields high-quality mass spectra [46], it is technically cumbersome; direct on-plate testing using formic acid identies a similar number of organisms [37]. Direct on-plate testing must be avoided with organisms hazardous to microbiology technologists (eg, Brucella species) [28]. Reproduced by permission of Mayo Foundation for Medical Education and Research. All rights reserved. MEDICAL MICROBIOLOGY CID 2013:57 (15 August) 565
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
spectra to determine the relatedness of the spectrum to spectra in the database; a list of most closely related organisms is gener- ated, each with a numeric ranking (percentage or score) indicat- ing the level of condence in identication. Depending on how high the value is, the organism is identied at the family, genus, or species level. As with any identication system, well-curated spectral databases representing comprehensive collections of correctly named and clinically relevant bacteria and fungi are necessary. In the absence of mass spectral entries in a database, entries may be manually added (assuming availability of requi- site isolates). The time from putting the target plate into the in- strument to nal result is fast; for example, the Bruker system produces results in 3 minutes per isolate. No mass spectrome- try expertise is required, and the technology is green with re- usable target plates available and few disposables. Commercial MALDI-TOF MS systems are available from Bruker Daltonics Inc (Billerica, Massachusetts) and bioMrieux Inc. A system used primarily in France (Andromas, Paris, France), will not be further discussed. The Bruker Biotyper system is most well studied and includes a mass spectrometer, software, and library; over the past few years, improved software and progressively more comprehensive and better curated mass spectral libraries have been made available. AnagnosTec (Zossen, Germany) previously marketed a microbial database called SARAMIS used with Shimadzus AXIMA Assurance mass spectrometer (Shimadzu, Columbia, Maryland), which was acquired by bioMrieux in 2010; with this acquisition, the systems name was changed to VITEK MS RUO. BioMrieux developed its own database/operating software/algorithms, re- ferred to as VITEK MS (including a prerelease version of the VITEK MS v1.1 database [6], the v1 system/v1.1 database [710], the v2.0 system/v2.0 database and algorithms, and VITEK MS Plus, which incorporates the VITEK MS v2.0 and SARAMIS v4.0 databases). Rapidly implemented progressive improve- ments in both systems are enhancing the clinical laboratorys ability to identify a range of organisms but render it challenging for those reading the literature to compare studies due to vari- able software, interpretive rules, databases, mass spectrometers, comparators, specimen preparations, organism sets (ie, en- riched for unusual organisms, or not), and so forth. Current systems databases are generally best for common aerobic gram-negative bacilli and gram-positive cocci, and may lack some esoteric organisms, such as certain anaerobes, fungi, and mycobacteria. Given changing nomenclature and ongoing de- scription of new species and emerging microorganisms, regular and ongoing updates to databases are imperative to ensure clin- ical utility. Databases should include entries representing major phylogenetic lineages within each species. It is possible for users to add their own mass spectral entries to enhance existing databases or create their own database by including locally im- portant strains or strains not well represented in commercial libraries. The Bruker and bioMrieux MALDI-TOF MS systems differ in databases, algorithms used to identify organisms, and instru- mentation. Numeric rankings are reported on different scales, and are not directly comparable. Brukers Microex LT mass spectrometer is a desktop instrument, whereas bioMrieuxs is a larger, oor model. At this time, neither system is approved by the US Food and Drug Administration. Figure 2. Matrix-assisted laser desorption ionizationtime of ight mass spectrometer. The target plate is placed into the ionization chamber of the mass spectrometer. Spots to be analyzed are shot by an ultraviolet N 2 laser desorbing microbial and matrix molecules from the target plate. The majority of energy is absorbed by the matrix, converting it to an ionized state. Through random collision in the gas phase, charge is trans- ferred from matrix to microbial molecules. The cloud of ionized molecules is funneled through a positively charged electrostatic eld into the time of ight mass analyzer, a tube under vacuum. The ions travel toward an ion detector with small analytes traveling fastest, followed by progressively larger analytes. As ions emerge from the mass analyzer, they collide with an ion detector generating a mass spectrum representing the number of ions hitting the detector over time. Although separation is by mass- to-charge ratio, because the charge is typically single for the described ap- plication, separation is effectively by molecular weight. Reproduced by permission of Mayo Foundation for Medical Education and Research. All rights reserved. 566 CID 2013:57 (15 August) MEDICAL MICROBIOLOGY
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
Generally speaking, MALDI-TOF MS performs at least as well as, if not better than, automated biochemical identication for commonly encountered bacteria and yeast (Table 1), and better for unusual organisms. We compared the BD Phoenix Automated Microbiology System and Biotyper system using 440 common and unusual gram-negative bacilli; the former correctly identied 83% and 75% of isolates to the genus and species levels, respectively, whereas MALDI-TOF MS correctly identied 93% and 82% of isolates to the genus and species levels, respectively [11]. We also evaluated the Biotyper system using 217 clinical isolates of staphylococci, streptococci, and enterococci, 98% and 79% of which were correctly identied to the genus and species levels, respectively [12]. The performance of the 2 commercial systems appears to be similar. Martiny et al reported 93% species-level identications of 1003 routine bac- terial isolates with both a prerelease version of the VITEK MS v1.1 database and the Biotyper system [6]. In most cases, organisms are either correctly identied or yield a low score/percentage, indicating that identication has not been achieved; the latter typically implies no match in the database but can occur due to technically poor preparation. Misidentications are unusual but may occur with some closely related organisms. Although a comprehensive review of identi- cation of specic organisms is beyond the scope of this article, some generalizations can be made. Identication of staphylo- cocci [13] and -hemolytic streptococci is excellent [14]. The -hemolytic streptococci are more problematic because Strepto- coccus mitis and Streptococcus oralis may be poorly differentiat- ed from one another and from Streptococcus pneumoniae, at least with the Bruker system [12, 15]; recent data suggest that VITEK MS may overcome this limitation [8]. Arcanobacterium haemolyticum, Rhodococcus equi, and all but select closely related Corynebacterium species are reliably identied [9, 16]. Some closely related organisms, such as Escherichia coli and Shigella species, are not differentiated. (To avoid potential mis- identication, we attempt to avoid testing of suspect E. coli and we perform additional testing if E. coli is suggested.) MALDI- TOF MS accurately detects Enterobacter cloacae complex, although it may not discriminate some species within this complex [9, 17]. Likewise, accurate differentiation of all species of Acinetobacter may be challenging with current databases, al- though Acinetobacter baumannii is identiable [18]. Similar challenges exist with other identication systems. MALDI-TOF MS is exciting because of the breadth and depth of organism types that can be identied with one platform, including, but not limited to, Aeromonas species [19], Campylobacter species, Arcobacter butzleri, and Yersinia enterocolitica [20]. MALDI- TOF MS can identify and differentiate Capnocytophaga cani- morsus from Capnocytophaga cynodegmi [21]. Although typing within the genus Salmonella is not generally possible, it may be possible to distinguish Salmonella enterica subspecies enterica serovar Typhi from other S. enterica serovars [22]. And with user-supplemented databases, HACEK organisms [23], Legion- ella species [24], and Nocardia species [25] (the last with specif- ic extraction procedures) may be identied. Jamal et al reported species-level identication of 89% and 100% of 274 routinely isolated anaerobic bacteria (enriched in Bacteroides fragilis) using the Biotyper DB Update v3.3 and the VITEK MS v1 system/v1.1 database, respectively [10]. Identi- cation of more esoteric anaerobes, including Prevotella species, has been successful in 83% of cases with user supplementation of Brukers Reference Library 3.2.1.0 [26]. We evaluated a diverse collection of 253 clinical anaerobic isolates using the Bi- otyper system and a user-supplemented database; 92% and 71% of isolates were correctly identied to the genus and species levels, respectively [27]. Further commercial database Table 1. Examples of Recent Evaluations of Matrix-Assisted Laser Desorption IonizationTime of Flight Mass Spectrometry for Routine Bacterial Identication Isolates % Identification System No. Type Period of Isolate Collection Genus Level Species Level Country Comparator Reference Bruker Biotyper 1013 Bacteria 2 mo 99% 97% France Phoenix, API, Biochemical [72] Bruker Biotyper 468 Bacteria 3 mo 97% 92% Japan MicroScan, API, Phoenix [73] Bruker Biotyper 2781 Bacteria 1 mo 96% 85% Australia VITEK2, API, Biochemical [74] Vitek MS a 767 Bacteria 6 wk 95% 87% France VITEK2 [8] Bruker Biotyper/ Vitek MSb 986 Bacteria 3 mo 96%/94% 93%/93% Belgium Bruker Biotyper compared to Vitek MS [6] a v1 system/v1.1 database. b Prerelease version of v1.1 database. MEDICAL MICROBIOLOGY CID 2013:57 (15 August) 567
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
supplementation will improve anaerobic identication to a level previously possible only with 16S ribosomal RNA gene se- quencing or gas liquid chromatography. MALDI-TOF MS can accurately identify Francisella tularen- sis and Brucella species; however, the Biotyper library does not contain and will not identify these organisms. Use of Brukers Security Relevant database enables Brucella species and F. tularensis identication [28]. A handful of investigators have reported using MALDI-TOF MS for mycobacteria. This requires special processing to kill tested organisms (for safety), disrupt clumped cells, and break down cell envelopes. Current commercial libraries inadequately address the clinically relevant Mycobacterium species, but with appropriate library construction, MALDI-TOF MS should be able to identify most, with a few caveats expected. Members of the Mycobacterium tuberculosis complex are identied at the complex level [29]; some species (eg, Mycobacterium abscessus/ massiliense; Mycobacterium mucogenicum/phocaicum; Myco- bacterium chimaera/intracellulare) may not be well differentiat- ed from one another [29], and identication from liquid media may be more challenging than from colonies on solid media [30]. Beyond bacteria, MALDI-TOF MS can identify yeast, out- performing some conventional phenotypic systems, and distin- guishing Candida dubliniensis and albicans; Candida rugosa and pararugosa; Candida norvegensis, krusei, and inconspicua; Candida parapsilosis, orthopsilosis, and metapsilosis [31]; and (with database supplementation) Cryptococcus neoformans and gattii [3234]. Dhiman et al evaluated the Biotyper system for identication of 138 common and 103 unusual yeast isolates, and reported 96% and 85% accurate species-level identication, respectively [35]. MALDI-TOF MS may outperform current systems to identify esoteric and sometimes misidentied Candida species, such as Candida famata [31]. Although older studies used preparatory extraction for yeasts, we and others use the same direct testing strategy we use for bacteria (Figure 1) [36, 37]. Few lamentous fungi are included in current commercial databases. Filamentous fungi exhibit variable phenotypes; protein spectra may vary with growth conditions or with the zone of fungal mycelium analyzed. De Carolis et al developed a library of Aspergillus species, Fusarium species, and Mucorales using the Biotyper system and identied 97% of 94 isolates to the species level [38]. Using the VITEK MS v1 system/v1.1 da- tabase and direct on-plate testing, Iriart et al identied 82% of 44 Aspergillus isolates (including all isolates with species in the database) [7]. Theel et al developed a dermatophyte library and used it to identify 93% and 60% of 171 dermatophyte isolates to the genus and species levels, respectively, using the Biotyper system [39]. Preliminary studies indicate that Pseudallescheria/ Scedosporium complex species are identiable [40]. Beyond fungi, MALDI-TOF MS has been used for Prototheca species identication [41]. Compared to standard methods, MALDI-TOF MS improves turn-around time for bacterial and fungal identication by an average of 1.45 days [42], and, because only a small amount of organism is required, testing can be performed directly from single colonies on primary culture plates, whereas other methods may require subculture. Tests for screening for some enteric pathogens (eg, triple sugar iron agar for Salmonella species) may be eliminated [43]. MALDI-TOF MS reagent costs are approximately $0.35/test; implementation reduces reagent and labor costs for identica- tion, in one model, by 57% within 1 year [42]. An estimated 87% of isolates are identied on the rst day (compared with 9% with standard techniques), with nal identications a day earlier for most organisms, and several days earlier for bio- chemically inert, fastidious, or slow-growing organisms [42]. DNA sequencing expenses are avoided for some esoteric organ- isms, waste disposal is decreased, and quality control and labo- ratory technologist training/labor for replaced/retired tests are eliminated [44]. Adoption of MALDI-TOF MS impacts laboratory workow [9]. Time at the bench assessing primary cultures is relatively unaf- fected, with subcultures needed for susceptibility testing. Gram staining of colonies may not always be needed, as this informa- tion is not required for MALDI-TOF MS. For some organisms (eg, S. aureus), rapid tests performed at the bench will remain. MALDI-TOF MS has limitations (Table 2). Unlike publically available sequence databases such as GenBank, MALDI-TOF MS databases are proprietary. Although low identication per- centages for some organisms may be improved by user addition of mass spectral entries of underrepresented species or strains (to cover intraspecies variability) to the database, or even read- dition of reference strain spectra [26], doing so may be beyond the capabilities of some laboratories. Because of low scores/per- centages, repeat testing is required for approximately 10% of isolates [42]. Growth on some media (eg, colistin-nalidixic acid agar) may be associated with low scores/percentages [45], and tiny or mucoid colonies may fail identication [9, 46]. Rened criteria are needed to distinguish closely related species and to differentiate them from the next best taxon match [47]. For certain species of organisms, genus- or species-specic (includ- ing lowered) cutoffs may be appropriate [13, 16, 48]. Laboratory errors may occur, including colony inoculation in erroneous target plate locations, testing impure colonies, smearing be- tween spots, failure to clean target plates, and erroneous result entry into laboratory information systems. There is a learning curve to applying ideal colony amounts to target plates [9]. In- strument cost is comparable to that of other common laborato- ry equipment; laser, software, hardware, or mass spectrometer failure require an appropriate service plan. Although MALDI- TOF MS is generally reproducible, sources of variability in- clude the mass spectrometer, matrix and solvent composition, 568 CID 2013:57 (15 August) MEDICAL MICROBIOLOGY
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
technologists, culture conditions, and biological variability; quality control strategies are being dened. Notably, MALDI- TOF MS may yield different (generally more accurate) identi- cation than current systems [11, 49]. Antimicrobial susceptibility is not directly determined with the above-described strategy. By rapid organism identication, intrinsic antimicrobial resistance characteristics of particular species (or typical susceptibility of the identied species based on local antibiograms) may guide therapy. Some investigators have incubated bacteria with antibiotics and measured antibiot- ic degradation using MALDI-TOF MS; this requires specic system conguration and incubation (ie, to allow antibiotic hy- drolysis) and applies only to resistance mechanisms associated with antibiotic degradation (eg, -lactamases) [50]. Although detection of methicillin resistance in S. aureus has been contro- versial, this does not appear to be feasible using the Biotyper software in detection ranges and instrument settings preset for microbial identication [51]. Microbial strain typing is under development with no current universal protocol; innovations and advances are expected. With strain typing [52, 53], strains of S. aureus likely to be methicillin resistant (eg, USA300 [54]), en- terococci likely to be vancomycin resistant [55], or Bacteroides fragilis likely to be carbapenem resistant (cA positive) [56] may be called out (as markers for possible resistance). Although direct testing of clinical specimens is not generally feasible because of a high limit of detection, urine may be tested directly due to the high numbers of bacteria present in clinically infected urine (although -defensins 1, 2, and 3 may impair database matching [57]). Rapid identication of microorganisms growing in blood culture bottles is promising, but requires preparatory specimen processing as blood culture bottles contain macromolecules from blood and growth media. Processing is accomplished with differential centrifugation and washings, selective lysis of blood cells (eg, using water, ammonium chloride, saponin, sodium dodecyl sulfate), separator gels, ltration, and so on; commer- cial blood culture bottle processing using the Sepsityper (Bruker Daltonics) is available [5864]. Although results are valid when obtained, the yield is generally not as good as direct colony testing with a higher percentage of gram-negative than gram-positive organisms being identied [58]; polymicrobial cultures may not be fully characterized (although strategies are being developed). Most Candida species are identiable [65]. An alternate approach is to subculture positive blood culture bottles to solid media and test growth after a short period (eg, 24 hours) of incubation [66, 67]. By testing of positive blood culture bottles using MALDI-TOF MS, the time to organism identication may be reduced by a day or more, which may serve as a guide for empirical treatment [68], but antimicrobial susceptibility is not provided. Recently, MALDI-TOF MS has been tied with direct antimicrobial susceptibility testing from positive blood culture bottles [69, 70], improving time to optimal therapy and decreasing hospital length of stay and costs [71]. In summary, MALDI-TOF MS is a quantum leap forward for rapid and accurate identication of cultured bacteria and fungi in clinical microbiology. This is incontrovertibly bene- cial to laboratories, and decreased expense is helpful to labora- tories and patients alike. MALDI-TOF MS implementation may impact patient outcomes and/or cost of care, depending on its application. Further, this new technology may provide better denition of the epidemiology, pathogenesis, and antimi- crobial susceptibility of hitherto unrecognized (or misidenti- ed) microorganisms. Notes Acknowledgments. I thank Dr Nancy L. Wengenack, Scott A. Cun- ningham, Brenda L. Dylla, and Sherry M. Ihde for their thoughtful reviews of this manuscript. Potential conicts of interest. Author certies no potential conicts of interest. The author has submitted the ICMJE Form for Disclosure of Potential Conicts of Interest. Conicts that the editors consider relevant to the content of the manuscript have been disclosed. Table 2. Advantages and Limitations of Matrix-Assisted Laser Desorption IonizationTime of Flight Mass Spectrometry in Clini- cal Microbiology Advantages Limitations Rapid turn-around time Does not provide antimicrobial susceptibility Single colony requirement Requires isolated colony Automated, high throughput Repeat testing occasionally required Broad applicability to bacteria and fungi, including esoteric organisms Not cleared by the US Food and Drug Administration Databases may be expanded by users Identification limited by entries in database; databases vary, not publicly available Minimal consumables Laser, hardware, software, mass spectrometer failure potential testing downtime Cost-effective Instrument cost Generalizes to bacteria and yeast Misidentifications: failure to test pure colonies or to clean target plates Some closely related organisms not differentiated (eg, Escherichia coli and Shigella species) Growth on some media associated with no identification (without extraction) Tiny or mucoid colonies may not be identified MEDICAL MICROBIOLOGY CID 2013:57 (15 August) 569
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
References 1. Seng P, Drancourt M, Gouriet F, et al. Ongoing revolution in bacteriol- ogy: routine identication of bacteria by matrix-assisted laser desorp- tion ionization time-of-ight mass spectrometry. Clin Infect Dis 2009; 49:54351. 2. Anhalt JP, Fenselau C. Identication of bacteria using mass spectrome- try. Anal Chem 1975; 47:21925. 3. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 1988; 60:2299301. 4. Tanaka K, Hiroaki W, Ido Y, Akita S, Yoshida Y, Yoshida T. Protein and polymer analyses up to m/z 100 000 by laser ionization time- of-ight mass spectrometry. Rapid Commun Mass Spectrom 1988; 2:1513. 5. Mellmann A, Cloud J, Maier T, et al. Evaluation of matrix-assisted laser desorption ionization-time-of-ight mass spectrometry in comparison to 16S rRNA gene sequencing for species identication of nonferment- ing bacteria. J Clin Microbiol 2008; 46:194654. 6. Martiny D, Busson L, Wybo I, El Haj RA, Dediste A, Vandenberg O. Comparison of the Microex LT and Vitek MS systems for routine identication of bacteria by matrix-assisted laser desorption ionization- time of ight mass spectrometry. J Clin Microbiol 2012; 50:131325. 7. Iriart X, Lavergne RA, Fillaux J, et al. Routine identication of medical fungi by the new Vitek MS matrix-assisted laser desorption ionization- time of ight system with a new time-effective strategy. J Clin Microbiol 2012; 50:210710. 8. Dubois D, Grare M, Prere MF, Segonds C, Marty N, Oswald E. Perfor- mances of the Vitek MS matrix-assisted laser desorption ionization-time of ight mass spectrometry system for rapid identication of bacteria in routine clinical microbiology. J Clin Microbiol 2012; 50:256876. 9. Harris P, Winney I, Ashhurst-Smith C, OBrien M, Graves S. Compari- son of Vitek MS (MALDI-TOF) to standard routine identication methods: an advance but no panacea. Pathology 2012; 44:5835. 10. Jamal WY, Shahin M, Rotimi VO. Comparison of two matrix-assisted laser desorption ionization-time of ight (MALDI-TOF) mass spec- trometry methods and API 20AN for identication of clinically rele- vant anaerobic bacteria. J Med Microbiol 2013; 62:5404. 11. Saffert RT, Cunningham SA, Ihde SM, Jobe KE, Mandrekar J, Patel R. Comparison of Bruker Biotyper matrix-assisted laser desorption ioniza- tion-time of ight mass spectrometer to BD Phoenix automated micro- biology system for identication of gram-negative bacilli. J Clin Microbiol 2011; 49:88792. 12. Alatoom AA, Cunningham SA, Ihde SM, Mandrekar J, Patel R. Com- parison of direct colony method versus extraction method for identi- cation of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of ight mass spectrometry. J Clin Mi- crobiol 2011; 49:286873. 13. Richter C, Hollstein S, Woloszyn J, Kaase M, Gatermann SG, Szabados F. Evaluation of species-specic score cut-off values for various Staphy- lococcus species using a MALDI Biotyper-based identication. J Med Microbiol 2012; 61:140916. 14. Cherkaoui A, Emonet S, Fernandez J, Schorderet D, Schrenzel J. Evalu- ation of matrix-assisted laser desorption ionization-time of ight mass spectrometry for rapid identication of beta-hemolytic streptococci. J Clin Microbiol 2011; 49:30045. 15. Davies AP, Reid M, Hadeld SJ, et al. Identication of clinical isolates of alpha-hemolytic streptococci by 16S rRNA gene sequencing, matrix- assisted laser desorption ionization-time of ight mass spectrometry using MALDI Biotyper, and conventional phenotypic methods: a com- parison. J Clin Microbiol 2012; 50:408790. 16. Alatoom AA, Cazanave CJ, Cunningham SA, Ihde SM, Patel R. Identi- cation of non-diphtheriae Corynebacterium by use of matrix-assisted laser desorption ionization-time of ight mass spectrometry. J Clin Mi- crobiol 2012; 50:1603. 17. Pavlovic M, Konrad R, Iwobi AN, Sing A, Busch U, Huber I. A dual ap- proach employing MALDI-TOF MS and real-time PCR for fast species identication within the Enterobacter cloacae complex. FEMS Micro- biol Lett 2012; 328:4653. 18. Alvarez-Buylla A, Culebras E, Picazo JJ. Identication of Acinetobacter species: is Bruker Biotyper MALDI-TOF mass spectrometry a good al- ternative to molecular techniques? Infect Genet Evol 2012; 12:3459. 19. Lamy B, Kodjo A, Laurent F. Identication of Aeromonas isolates by matrix-assisted laser desorption ionization time-of-ight mass spec- trometry. Diagn Microbiol Infect Dis 2011; 71:15. 20. He Y, Li H, Lu X, Stratton CW, Tang YW. Mass spectrometry biotyper system identies enteric bacterial pathogens directly from colonies grown on selective stool culture media. J Clin Microbiol 2010; 48: 388892. 21. Zangenah S, Ozenci V, Borang S, Bergman P. Identication of blood and wound isolates of C. canimorsus and C. cynodegmi using VITEK2 and MALDI-TOF. Eur J Clin Microbiol Infect Dis 2012; 31:26317. 22. Kuhns M, Zautner AE, Rabsch W, et al. Rapid discrimination of Salmo- nella enterica serovar Typhi from other serovars by MALDI-TOF mass spectrometry. PLoS One 2012; 7:e40004. 23. Couturier MR, Mehinovic E, Croft AC, Fisher MA. Identication of HACEK clinical isolates by matrix-assisted laser desorption ionization- time of ight mass spectrometry. J Clin Microbiol 2011; 49:11046. 24. Gaia V, Casati S, Tonolla M. Rapid identication of Legionella spp. by MALDI-TOF MS based protein mass ngerprinting. Syst Appl Micro- biol 2011; 34:404. 25. Verroken A, Janssens M, Berhin C, et al. Evaluation of matrix-assisted laser desorption ionization-time of ight mass spectrometry for identi- cation of Nocardia species. J Clin Microbiol 2010; 48:401521. 26. Wybo I, Soetens O, De Bel A, et al. Species identication of clinical Pre- votella isolates by matrix-assisted laser desorption ionization-time of ight mass spectrometry. J Clin Microbiol 2012; 50:14158. 27. Schmitt BH, Cunningham SA, Dailey AL, Gustafson DR, Patel R. Iden- tication of anaerobic bacteria by Bruker Biotyper matrix assisted laser desorption ionization-time of ight mass spectrometry with on-plate formic acid preparation. J Clin Microbiol; 2013; 51:7826. 28. Cunningham SA, Patel R. Importance of using Brukers Security Rele- vant Library for Biotyper identication of Burkholderia pseudomallei, Brucella species and Francisella tularensis. J Clin Microbiol 2013; 51:163940. 29. Saleeb PG, Drake SK, Murray PR, Zelazny AM. Identication of myco- bacteria in solid-culture media by matrix-assisted laser desorption ioni- zation-time of ight mass spectrometry. J Clin Microbiol 2011; 49:17904. 30. Bille E, Dauphin B, Leto J, et al. MALDI-TOF MS Andromas strategy for the routine identication of bacteria, mycobacteria, yeasts, Aspergil- lus spp. and positive blood cultures. Clin Microbiol Infect 2012; 18:111725. 31. Castanheira M, Woosley LN, Diekema DJ, Jones RN, Pfaller MA. Candida guilliermondii and other species of Candida misidentied as Candida famata: assessment by Vitek 2, DNA sequencing analysis, and matrix-assisted laser desorption ionization-time of ight mass spec- trometry in two global antifungal surveillance programs. J Clin Micro- biol 2013; 51:11724. 32. Firacative C, Trilles L, Meyer W. MALDI-TOF MS enables the rapid identication of the major molecular types within the Cryptococcus neoformans/C. gattii species complex. PLoS One 2012; 7:e37566. 33. Sendid B, Ducoroy P, Francois N, et al. Evaluation of MALDI-TOF mass spectrometry for the identication of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals. Med Mycol 2013; 51:2532. 34. McTaggart LR, Lei E, Richardson SE, Hoang L, Fothergill A, Zhang SX. Rapid identication of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of ight mass spectrometry. J Clin Microbiol 2011; 49:30503. 570 CID 2013:57 (15 August) MEDICAL MICROBIOLOGY
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
35. Dhiman N, Hall L, Wohlel SL, Buckwalter SP, Wengenack NL. Perfor- mance and cost analysis of matrix-assisted laser desorption ionization- time of ight mass spectrometry for routine identication of yeast. J Clin Microbiol 2011; 49:16146. 36. Van Herendael BH, Bruynseels P, Bensaid M, et al. Validation of a modied algorithm for the identication of yeast isolates using matrix- assisted laser desorption/ionisation time-of-ight mass spectrometry (MALDI-TOF MS). Eur J Clin Microbiol Infect Dis 2012; 31:8418. 37. Theel ES, Schmitt BH, Hall L, et al. Formic acid-based direct, on-plate testing of yeast and Corynebacterium species by Bruker Biotyper matrix-assisted laser desorption ionization-time of ight mass spec- trometry. J Clin Microbiol 2012; 50:30935. 38. De Carolis E, Posteraro B, Lass-Florl C, et al. Species identication of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-ight mass spec- trometry. Clin Microbiol Infect 2012; 18:47584. 39. Theel ES, Hall L, Mandrekar J, Wengenack NL. Dermatophyte identi- cation using matrix-assisted laser desorption ionization-time of ight mass spectrometry. J Clin Microbiol 2011; 49:406771. 40. Coulibaly O, Marinach-Patrice C, Cassagne C, Piarroux R, Mazier D, Ranque S. Pseudallescheria/Scedosporium complex species identica- tion by matrix-assisted laser desorption ionization time-of-ight mass spectrometry. Med Mycol 2011; 49:6216. 41. Murugaiyan J, Ahrholdt J, Kowbel V, Roesler U. Establishment of a matrix-assisted laser desorption ionization time-of-ight mass spec- trometry database for rapid identication of infectious achlorophyllous green micro-algae of the genus Prototheca. Clin Microbiol Infect 2012; 18:4617. 42. Tan KE, Ellis BC, Lee R, Stamper PD, Zhang SX, Carroll KC. Prospec- tive evaluation of a matrix-assisted laser desorption ionization-time of ight mass spectrometry system in a hospital clinical microbiology lab- oratory for identication of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identication and cost-effec- tiveness. J Clin Microbiol 2012; 50:33018. 43. Sparbier K, Weller U, Boogen C, Kostrzewa M. Rapid detection of Sal- monella sp. by means of a combination of selective enrichment broth and MALDI-TOF MS. Eur J Clin Microbiol Infect Dis 2012; 31:76773. 44. Gaillot O, Blondiaux N, Loiez C, et al. Cost-effectiveness of switch to matrix-assisted laser desorption ionization-time of ight mass spec- trometry for routine bacterial identication. J Clin Microbiol 2011; 49:4412. 45. Anderson NW, Buchan BW, Riebe KM, Parsons LN, Gnacinski S, Lede- boer NA. Effects of solid-medium type on routine identication of bac- terial isolates by use of matrix-assisted laser desorption ionization-time of ight mass spectrometry. J Clin Microbiol 2012; 50:100813. 46. Xiao D, Zhao F, Lv M, et al. Rapid identication of microorganisms iso- lated from throat swab specimens of community-acquired pneumonia patients by two MALDI-TOF MS systems. Diagn Microbiol Infect Dis 2012; 73:3017. 47. De Bel A, Wybo I, Vandoorslaer K, Rosseel P, Lauwers S, Pierard D. Acceptance criteria for identication results of gram-negative rods by mass spectrometry. J Med Microbiol 2011; 60:6846. 48. Szabados F, Tix H, Anders A, Kaase M, Gatermann SG, Geis G. Evalua- tion of species-specic score cutoff values of routinely isolated clinically relevant bacteria using a direct smear preparation for matrix-assisted laser desorption/ionization time-of-ight mass spectrometry-based bac- terial identication. Eur J Clin Microbiol Infect Dis 2012; 31:110919. 49. Dupont C, Sivadon-Tardy V, Bille E, et al. Identication of clinical co- agulase-negative staphylococci, isolated in microbiology laboratories, by matrix-assisted laser desorption/ionization-time of ight mass spec- trometry and two automated systems. Clin Microbiol Infect 2010; 16:9981004. 50. Sparbier K, Schubert S, Weller U, Boogen C, Kostrzewa M. Matrix- assisted laser desorption ionization-time of ight mass spectrometry- based functional assay for rapid detection of resistance against beta- lactam antibiotics. J Clin Microbiol 2012; 50:92737. 51. Szabados F, Kaase M, Anders A, Gatermann SG. Identical MALDI TOF MS-derived peak proles in a pair of isogenic SCCmec-harboring and SCCmec-lacking strains of Staphylococcus aureus. J Infect 2012; 65:4005. 52. Wolters M, Rohde H, Maier T, et al. MALDI-TOF MS ngerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages. Int J Med Microbiol 2011; 301:648. 53. Mencacci A, Monari C, Leli C, et al. Identication of nosocomial out- breaks of Acinetobacter baumannii using MALDI-TOF mass spectrom- etry. J Clin Microbiol 2013; 51:6036. 54. Boggs SR, Cazares LH, Drake R. Characterization of a Staphylococcus aureus USA300 protein signature using matrix-assisted laser desorp- tion/ionization time-of-ight mass spectrometry. J Med Microbiol 2012; 61:6404. 55. Grifn PM, Price GR, Schooneveldt JM, et al. Use of matrix-assisted laser desorption ionization-time of ight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak. J Clin Microbiol 2012; 50:291831. 56. Wybo I, De Bel A, Soetens O, et al. Differentiation of cA-negative and cA-positive Bacteroides fragilis isolates by matrix-assisted laser desorp- tion ionization-time of ight mass spectrometry. J Clin Microbiol 2011; 49:19614. 57. Kohling HL, Bittner A, Muller KD, et al. Direct identication of bacte- ria in urine samples by matrix-assisted laser desorption/ionization time-of-ight mass spectrometry and relevance of defensins as interfer- ing factors. J Med Microbiol 2012; 61:33944. 58. Buchan BW, Riebe KM, Ledeboer NA. Comparison of the MALDI Bio- typer system using Sepsityper specimen processing to routine microbi- ological methods for identication of bacteria from positive blood culture bottles. J Clin Microbiol 2012; 50:34652. 59. Meex C, Neuville F, Descy J, et al. Direct identication of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction. J Med Microbiol 2012; 61:15116. 60. Martiny D, Dediste A, Vandenberg O. Comparison of an in-house method and the commercial Sepsityper kit for bacterial identication directly from positive blood culture broths by matrix-assisted laser de- sorption-ionisation time-of-ight mass spectrometry. Eur J Clin Micro- biol Infect Dis 2012; 31:226981. 61. Loonen AJ, Jansz AR, Stalpers J, Wolffs PF, van den Brule AJ. An evalu- ation of three processing methods and the effect of reduced culture times for faster direct identication of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS. Eur J Clin Microbiol Infect Dis 2012; 31:157583. 62. Saffert RT, Cunningham SA, Mandrekar J, Patel R. Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry. Diagn Microbiol Infect Dis 2012; 73:216. 63. Fothergill A, Kasinathan V, Hyman J, Walsh J, Drake T, Wang YF. Rapid identication of bacteria and yeasts from positive BacT/ALERT blood culture bottles by using a lysis-ltration method and MALDI- TOF mass spectrum analysis with SARAMIS database. J Clin Microbiol 2013: 51:8059. 64. Drancourt M. Detection of microorganisms in blood specimens using matrix-assisted laser desorption ionization time-of-ight mass spec- trometry: a review. Clin Microbiol Infect 2010; 16:16205. 65. Spanu T, Posteraro B, Fiori B, et al. Direct MALDI-TOF mass spec- trometry assay of blood culture broths for rapid identication of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories. J Clin Microbiol 2012; 50: 1769. 66. Ngan GJ, Lin RT, Teo JW. Utility of the Bruker Biotyper matrix-assisted laser desorption ionisation time-of-ight mass spectrometer in a clini- cal microbiology laboratory. Pathology 2012; 44:4936. MEDICAL MICROBIOLOGY CID 2013:57 (15 August) 571
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d
f r o m
67. Kroumova V, Gobbato E, Basso E, Mucedola L, Giani T, Fortina G. Direct identication of bacteria in blood culture by matrix-assisted laser desorption/ionization time-of-ight mass spectrometry: a new methodological approach. Rapid Commun Mass Spectrom 2011; 25:22479. 68. Clerc O, Prodhom G, Vogne C, Bizzini A, Calandra T, Greub G. Impact of matrix-assisted laser desorption ionization time-of-ight mass spectrometry on the clinical management of patients with gram- negative bacteremia: a prospective observational study. Clin Infect Dis 2013; 56:11017. 69. Romero-Gomez MP, Gomez-Gil R, Pano-Pardo JR, Mingorance J. Identication and susceptibility testing of microorganism by direct inoculation from positive blood culture bottles by combining MALDI- TOF and Vitek-2 Compact is rapid and effective. J Infect 2012; 65:51320. 70. Wimmer JL, Long SW, Cernoch P, et al. Strategy for rapid identication and antibiotic susceptibility testing of gram-negative bacteria directly recovered from positive blood cultures using the Bruker MALDI Bio- typer and the BD Phoenix system. J Clin Microbiol 2012; 50:24524. 71. Perez K, Olsen R, Musick W, et al. Integrating rapid pathogen identi- cation and antimicrobial stewardship signicantly decreases hospital costs [Epub ahead of print]. Arch Pathol Lab Med. 2013; doi:10.5858/ arpa.2012-0651-0A. 72. Bessde E, Angla-Gre M, Delagarde Y, Sep Hieng S, Mnard A, Mgraud F. Matrix-assisted laser-desorption/ionization biotyper: expe- rience in the routine of a university hospital. Clin Microbiol Infect 2011; 17:5338. 73. Sogawa K, Watanabe M, Sato K, et al. Use of the MALDI BioTyper system with MALDI-TOF mass spectrometry for rapid identication of microorganisms. Anal Bioanal Chem 2011; 400:190511. 74. Neville SA, Lecordier A, Ziochos H, et al. Utility of matrix-assisted laser desorption ionization-time of ight mass spectrometry following introduction for routine laboratory bacterial identication. J Clin Mi- crobiol 2011; 49:29804. 572 CID 2013:57 (15 August) MEDICAL MICROBIOLOGY
b y
g u e s t
o n
A u g u s t
2 3 ,
2 0 1 4 h t t p : / / c i d . o x f o r d j o u r n a l s . o r g / D o w n l o a d e d