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Multicenter Study Evaluating the Vitek MS
System for Identification of Medically
Important Yeasts

Lars F. Westblade, Rebecca Jennemann, John A. Branda,
Maureen Bythrow, Mary Jane Ferraro, Omai B. Garner,
Christine C. Ginocchio, Michael A. Lewinski, Ryhana Manji,

A. Brian Mochon, Gary W. Procop, Sandra S. Richter,
A. Rychert, Linda Sercia and Carey-Ann D. Burnham

2013, 51(7):2267. DOI:
J. Clin. Microbiol.
Published Ahead of Print 8 May 2013.
Updated information and services can be found at: These include: This article cites 35 articles, 17 of which can be accessed freeat: Receive: RSS Feeds, eTOCs, free email alerts (when newarticles cite this article), Information about commercial reprint orders: To subscribe to to another ASM Journal go to:

Multicenter Study Evaluating the Vitek MS System for Identification
of Medically Important Yeasts

Lars F. Westblade,a,b Rebecca Jennemann,c John A. Branda,d Maureen Bythrow,e Mary Jane Ferraro,d Omai B. Garner,f
Christine C. Ginocchio,b,e Michael A. Lewinski,f Ryhana Manji,e A. Brian Mochon,f Gary W. Procop,g Sandra S. Richter,g
Jenna A. Rychert,d Linda Sercia,g Carey-Ann D. Burnhama

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USAa; Department of Pathology and Laboratory Medicine, Hofstra North Shore-LIJ School of Medicine, Hempstead, New York, USAb; Barnes-Jewish Hospital, St. Louis, Missouri, USAc; Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USAd; Department of Pathology and Laboratory Medicine, North Shore-LIJ Health System Laboratories, Lake Success, New York, USAe; Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USAf; Department of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio, USAg The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption
ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from
clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identify-
ing medically important yeasts. A collection of 852 isolates was tested, including 20 Candida
species (626 isolates, including 58 C.
, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast
isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid
overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold

standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were iden-
tified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identi-
fied, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans
(n 58) identified as
Candida dubliniensis
, one isolate of Candida parapsilosis (n 73) identified as Candida pelliculosa, and three isolates of Geotri-
chum klebahnii
(n 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior
to the phenotypic identification systems currently employed in clinical microbiology laboratories.

Asthenumberofpatientswithprofoundimmunosuppression matrix-assisted laser desorption ionization–time of flight
(such as those with solid-organ and hematopoietic stem cell (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS-based transplants) continues to rise, the morbidity and mortality bur- microbial identification relies on the generation of an organism- dens attributed to invasive fungal infections are increasing specific mass spectrum or "protein fingerprint" that is examined In the case of invasive fungal infections, expedient identification against a reference database to provide an organism identification of the offending organism is essential for optimal patient manage- The objective of this multicenter study was to assess the ment and the best clinical outcomes. As the antifungal suscepti- performance of the Vitek MS MALDI-TOF mass spectrometer bility profiles for many fungi (both yeasts and molds) are predict- (bioMérieux) in conjunction with the Vitek MS v2.0 database for able, organism identification frequently is sufficient to expedite the identification of yeasts isolated in diagnostic clinical microbi- appropriate empirical antifungal therapy. This has been demon- ology laboratories.
strated both to reduce the overall length of hospitalization and to (This work was presented in part as an abstract at the 113th maximize favorable clinical outcomes Conversely, the General Meeting of the American Society for Microbiology, Den- rapid exclusion of overt pathogenic or intrinsically resistant spe- ver, CO, 18 to 21 May 2013.) cies can be used to narrow therapy and/or to prevent treatment MATERIALS AND METHODS
with potentially toxic antifungal agents, thereby reducing negativeclinical outcomes and costs.
Isolates used in this study. Yeasts isolated and identified from clinical
specimens obtained from five diagnostic clinical microbiology laborato-
The methods for identification of yeasts in the diagnostic clin- ries, located at geographically distinct sites in North America, were in- ical microbiology laboratory have improved significantly over the cluded in the study. The study sites were Barnes-Jewish Hospital (St.
past several decades with methods ranging from simple Louis, MO), the Cleveland Clinic (Cleveland, OH), the UCLA Health manual biochemical assays to automated biochemical methods to System (Los Angeles, CA), the North Shore LIJ Core Laboratory (Lake sophisticated nucleic acid-based assays While these ad-vancements in methodology have greatly enhanced our ability toidentify yeasts, the limitations of these methods include cost, Received 11 March 2013 Returned for modification 26 March 2013 turnaround time, and, in some instances, the need for consid- Accepted 2 May 2013 erable expertise. Additionally, the accuracy of identification for Published ahead of print 8 May 2013 some less-common species is not optimal for some of the meth- Address correspondence to Carey-Ann D. Burnham,
Copyright 2013, American Society for Microbiology. All Rights Reserved.
A technology that is poised to revolutionize the rapid identifi- cation of yeasts isolated in the clinical microbiology laboratory is July 2013 Volume 51 Number 7 Journal of Clinical Microbiology Westblade et al.
TABLE 1 Performance characteristics of the Vitek MS system in identifying clinically relevant Candida species
No. (%) of isolates Identified correctly Identified correctly Candida albicans Candida famata Candida glabrata Candida kefyr Candida krusei Candida lambica Candida rugosa Candida utilis a Isolate misidentified as C. dubliniensis.
Isolate misidentified as C. pelliculosa. Success, NY), and the Massachusetts General Hospital (Boston, MA). In nexa, KS) twice before mass spectrometric analysis. Freshly collected iso- total, the collection tested was composed of 852 yeast isolates obtained lates were subcultured on SDA to assess purity before testing, or, if a pure from the five trial sites (508 isolates) and the bioMérieux stock collection culture was observed on the primary SDA plate, it was tested directly. All (344 isolates). The collection included 20 Candida species Cryp- isolates were analyzed within 72 h after visible growth at 35°C. In only four tococcus neoformans, and 10 species in the genera Geotrichum, Kodamaea, instances, isolates were taken from media other than SDA, including one Malassezia, Rhodotorula, Saccharomyces, and Trichosporon isolate taken from CHROMagar Candida (Becton, Dickinson, Sparks, Of the 344 isolates from the bioMérieux stock collection, 96 were used MD), one isolate taken from Mueller-Hinton II agar (Becton, Dickinson), in the development of the database. These isolates represent rare taxa, and two isolates taken from tryptic soy agar with sheep's blood (Remel).
such that it would not have been possible to evaluate them exclusively via In the four instances where SDA was not used to cultivate the strain for MS analysis, the MS identification matched the reference identification Cultivation of yeast isolates. The isolates were obtained from frozen
stocks or were tested fresh from clinical cultures. Strains that were stored Sample preparation. The yeast isolates were prepared for mass spec-
frozen were subcultured on Sabouraud dextrose agar (SDA; Remel, Le- trometric analysis using a direct, on-target, extraction method TABLE 2 Performance characteristics of the Vitek MS system in identifying clinically relevant non-Candida yeast species
No. (%) of isolates Identified correctly Identified correctly Kodamaea ohmeri Malassezia furfur a Isolates were misidentified as G. candidum. Journal of Clinical Microbiology Vitek MS Identification of Yeasts Briefly, a portion of a single colony was applied directly to a disposable isolates (2.8%) were not identified and five isolates (0.6%) were target slide (product no. 410893; bioMérieux, Marcy l'Etoile, France) composed of a polypropylene carrier with a stainless steel layer, using a Performance of the Vitek MS system in identifying Candida
1-␮l loop (product no. 861567010; Sarstedt, Newton, NC), and was lysed species. A total of 626 Candida isolates representing 20 different
by direct application of 0.5 ␮l formic acid (25% [vol/vol], product no.
species, including 58 Candida albicans, 62 C. glabrata, and 53 Can- 411072; bioMérieux) to the isolate immediately after application on the dida krusei isolates, were analyzed Of the 626 isolates, target plate. Immediately after the formic acid overlay was allowed to dry 616 (98.4%) were identified to the genus level and 612 (97.8%) at room temperature, 1 ␮l of matrix solution (3.1% [wt/vol] ␣-cyano-4- were identified to the species level. Only eight isolates (1.3%) were hydroxycinnamic acid, product no. 411071; bioMérieux) was applied andallowed to dry at room temperature prior to mass spectrometric analysis.
unidentified and two isolates (0.3%) were misidentified. The iso- Isolates were prepared for mass spectrometric analysis at the Vitek MS lates that were misidentified included one isolate of C. albicans preparation station, and the isolate information was transferred to the that was misidentified as Candida dubliniensis and one isolate of Vitek MS acquisition station using Myla v2.4 middleware. The total sam- Candida parapsilosis that was misidentified as Candida pelliculosa.
ple preparation time was approximately 1 min per isolate.
When the isolates from the bioMérieux stock collection were MALDI-TOF MS. Following sample preparation, samples were ana-
excluded, 16 species of Candida were represented. Of these 404 lyzed with the Vitek MS MALDI-TOF mass spectrometer in linear posi- isolates, 396 (98.0%) were identified correctly to the genus level tive-ion mode, across the mass-to-charge ratio range of 2,000 to 20,000 and 393 (97.3%) to the species level Da. Each spot was irradiated with 500 laser shots at 50 Hz. Target plates Performance of the Vitek MS system in identifying non-Can-
were calibrated and quality controlled both before and after data acquisi- dida yeast isolates. A total of 226 isolates representing 11 different
tion by using Escherichia coli ATCC 8739. Additionally, a Candida glabrata species, including 35 C. neoformans isolates, 50 Trichosporon iso- isolate (C. glabrata ATCC MYA-2950) and a sample containing matrixonly (negative control) were assayed for quality control purposes. After lates, and 35 Rhodotorula mucilaginosa isolates, were analyzed the acquisition of spectra, data were transferred from the Vitek MS acqui- The number of isolates identified to both the genus and sition station to the Vitek MS analysis server, and identification results species levels was 207 (91.6%), with all 35 (100%) C. neoformans were displayed using Myla v2.4 middleware. The total processing and data isolates correctly identified to the species level. The number of analysis time was approximately 20 min for a single isolate; this time isolates that were misidentified (three isolates [1.3%]) was low.
increased by approximately 1 min for each subsequent sample. Each op- The three misidentified isolates were Geotrichum klebahnii isolates erator participating in the study was required to analyze a proficiency that were identified as Geotrichum candidum. The proportion of panel successfully prior to beginning to test isolates for this investigation.
isolates that were not identified in this group (16 isolates [7.1%]) Data analysis. The Vitek MS identification system is based on com-
was greater than the proportion of isolates that were not identified parison of the characteristics of the spectra obtained with the Vitek MS in the Candida species group.
v2.0 database. This database was built using spectra for known strains for When the isolates from the bioMérieux stock collection were each claimed species. Based on this representative data collection, a weight is assigned to each peak for each species according to its specificity. As part excluded from this group of organisms, nine species of non-Can- of the identification process, the software compares the spectrum ob- dida yeast isolates remained. Of the 104 isolates, 99 (95.2%) were tained with peak weights defined for each claimed species. The resulting correctly identified to both the genus and species levels quantitative value, the confidence value, is calculated and expresses the Quality control. The C. glabrata quality control organism and
similarity between the unknown organism and every organism or organ- the negative control sample (matrix only) were tested by the Vitek ism group in the database. A single identification is displayed, with a MS every day that yeast isolates were assayed and with every new confidence value from 60.0 to 99.9, when one significant organism or lot of target slides, formic acid, and matrix. During the trial, the organism group is retained. "Low-discrimination" identifications are dis- quality control organism was tested 141 times and acceptable re- played when more than one but not more than four significant organisms sults were obtained 139 times (98.6%). Two quality control tests or organism groups are retained. In this case, the sum of confidence values yielded no identification upon initial testing. In both instances, is equal to 100. When more than four organisms or organism groups are however, the correct identification was obtained upon repeat test- found, or when no match is found, the organism is considered unidenti-fied.
ing on the same day. In all instances, the negative control yielded Molecular identification of yeast isolates. The molecular identifica-
no identification.
tion of all isolates in the test collection was performed by MIDI Labs(Newark, DE). The isolates were identified by sequencing the D2 region of the 26S rRNA gene using the MicroSeq D2 LSU rDNA fungal iden- Although the identification of yeast isolates has greatly improved tification kit (Applied Biosystems, Foster City, CA) Briefly, yeast over the past several decades, the manual and automated bio- genomic DNA was extracted and the D2 region was amplified by PCR; the chemical methods commonly used to identify contemporary yeast resultant PCR product was sequenced and compared with fungal se- isolates are time-consuming and may result in low-discrimination quences in the MicroSeq D2 fungal library and other public databases, identifications that require additional testing Nucleic ac- including GenBank id-based identification techniques, such as DNA sequencing ofyeast, have high accuracy but are expensive, might have prolonged turnaround times, and require technical expertise and equipment Overall performance of the Vitek MS system. A collection of 852
that may not be available to all laboratories. MALDI-TOF MS yeast isolates, comprising 31 different species obtained primarily offers a balance between speed and highly accurate yeast identifi- from clinical microbiology laboratories located in five different geographical regions in North America, was used to challenge the While fewer studies evaluating MALDI-TOF MS identification Vitek MS v2.0 database (bioMérieux). Of the 852 isolates included of yeasts than bacteria have been published to date, the theme of in the collection, 823 (96.6%) were identified to the genus level, the existing literature is that the performance of MALDI-TOF MS while 819 (96.1%) were identified to the species level. In total, 24 in identifying fungi, both yeasts and molds, is comparable or su- July 2013 Volume 51 Number 7 Westblade et al.
TABLE 3 Performance characteristics of the Vitek MS system in identifying Candida species recovered from clinical specimens
No. (%) of isolates Identified correctly Identified correctly Candida albicans Candida famata Candida glabrata Candida kefyr Candida krusei Candida lambica Candida rugosa a Isolate misidentified as C. dubliniensis. perior to that of conventional and nucleic acid-based identifica- ing the collection for strain heterogeneity. In addition, this study tion methods The major advantages of included a large number of isolates, and the identification of all MALDI-TOF MS identification of yeasts, compared with conven- isolates was verified using sequence analysis as a gold standard.
tional methods, are the marked decreases in cost and time to iden- Finally, this is the first study to date to evaluate the performance tification Antifungal susceptibility profiles generally are pre- characteristics of the Vitek MS v2.0 database for identification of dictable from the species identification and, of note, the four clinically relevant yeast species.
species of yeast that account for the vast majority of infections, i.e., The results of the multicenter study indicate that, independent C. albicans, C. glabrata, C. krusei, and C. parapsilosis, have distinct of the laboratory and the geographical origin of the isolates, the susceptibility profiles Therefore, rapid, highly accurate iden- Vitek MS demonstrated an overall species identification rate com- tification of yeast isolates using MALDI-TOF MS is poised to en- parable or superior to those for both traditional biochemical and hance patient care drastically and to reduce hospital-associated nucleic acid-based yeast identification systems but with a costs due to fungal infections.
significant reduction in the time to identification. This method is In this study, we evaluated the performance characteristics of technically facile and, once the laboratory has recovered the cap- the Vitek MS with the v2.0 database for identification of medically ital investment for the instrument purchase, the ongoing cost of important yeast species. This study has a number of strengths. The consumables is low.
first is that this was a multicenter evaluation; therefore, a large In our study, 24 (2.8%) and 5 (0.6%) isolates were not identi- number of independent operators were able to demonstrate the fied and were misidentified, respectively. Overall, we identified interlaboratory accuracy of this method. Isolates were recovered ⬎96% of the 852 isolates in this study to the species level. This is from geographically distinct areas across North America, enrich- comparable to the findings of other studies evaluating MALDI- TABLE 4 Performance characteristics of the Vitek MS system in identifying non-Candida yeast isolates recovered from clinical specimens
No. (%) of isolates Identified correctly Identified correctly Kodamaea ohmeri Malassezia furfur Journal of Clinical Microbiology Vitek MS Identification of Yeasts TOF MS identification of yeasts using other instrumentation plat- caution when using a direct plate extraction preparation method forms or spectral databases; Yaman and coworkers identified 94% is that the early growth of some thermally dimorphic fungi, such of 265 yeast isolates correctly using the Bruker Biotyper as Histoplasma capsulatum and Coccidioides immitis/posadasii, Bader and colleagues identified ⬎95% of 1,192 isolates correctly might resemble yeast-like colonies. Therefore, clinical laborato- using both the Bruker Biotyper and the Saramis instruments ries should be mindful of growth rates and colony morphology Dhiman and colleagues identified ⬎96% of 138 "common" yeasts when using this method for yeast identification.
and 84.5% of 103 "uncommon" yeasts to the species level using Despite the promising results reported in this study, there are the Bruker Biotyper and Iriart et al. identified 184 of 188 some limitations to our data. All except four of the isolates were yeast isolates (97.9%) tested using the Vitek MS In contrast grown on SDA for MALDI-TOF MS analysis; therefore, the per- to the current study, the study by Iriart et al. evaluated the formance characteristics of this methodology for yeast grown on Vitek MS v1.0 database and included primarily Candida isolates other types of media are unknown. For the 852 yeast isolates tested from a medical center in France, and sequencing was not the ref- in this study, all of the species identified are included in the Vitek erence method for the study.
MS v2.0 database. It is not known if unusual taxa not represented For the isolates that were misidentified in the current study, the in the database would be misidentified or simply not identified if incorrect identifications would be unlikely to lead to adverse clin- tested with this system. Finally, no isolates of Cryptococcus gattii, ical outcomes. Two of the five incorrectly identified isolates were an emerging fungal pathogen were included in the study.
Candida species, including an isolate of C. albicans misidentified Thus, the ability of the Vitek MS to differentiate C. neoformans as C. dubliniensis and an isolate of Candida parapsilosis misiden- from C. gattii, which might be of epidemiological and clinical tified as C. pelliculosa. The clinical impact of misidentifying C. importance, is not known. Previous studies using other platforms albicans as C. dubliniensis is likely to be minimal, although it has suggest that MALDI-TOF MS methods do have the potential for been suggested that the development of fluconazole resistance is species resolution of Cryptococcus species by permitting the addi- more likely for C. dubliniensis than for C. albicans C. parap- tion of mass spectra to the reference database The Vitek MS silosis exhibits higher MICs for the echinocandins than do most IVD system evaluated in this study does not permit user modifi- other Candida species therefore, misidentification might cations, such as the addition of spectra to the database.
be clinically significant. However, data on the susceptibility profile In conclusion, we present the results of a multicenter study of C. pelliculosa are sparse, and it is not obvious what empirical evaluating the Vitek MS system for identification of clinically rel- therapy might be initiated based on this identification. Although evant yeasts. Identification of yeasts using the Vitek MS is faster few isolates were not identified in this study, three (9.1%) of the and more accurate than phenotypic identification systems cur- Candida lusitaniae isolates tested were not identified. This is of rently employed in clinical microbiology laboratories and affords minor importance, compared with the overall performance char- accuracy comparable to that of more laborious and costly molec- acteristics of this method, but this finding is of note in light of the ular methods. Implementation of this methodology should fact that this species can be resistant to amphotericin B, a trait streamline yeast identification in the laboratory, positively affect unusual for Candida species patient care, and reduce health care-associated costs.
The three other misidentified isolates were Geotrichum klebah- nii identified as G. candidum. G. klebahnii is in the current data- base. While this error is unlikely to be clinically significant, bio- This study was funded by bioMérieux.
Mérieux indicated that future database and software updates will We thank Connie Bradford for her assistance with this study. We also result in reporting of these two species as G. candidum/klebahnii thank W. Michael Dunne, Jr., and Dave Pincus for their thoughtful re-views of the manuscript.
rather than specific species-level identification, to circumvent this J. A. Branda, J. A. Rychert, and M. J. Ferraro have received research misidentification event (bioMérieux, personal communication).
funding from bioMérieux and Becton, Dickinson and Co. C. C. Ginoc- In contrast to the "direct colony" methods typically used for chio has received research funding and consulting fees from bioMérieux MALDI-TOF MS identification of bacterial isolates, the majority and Becton, Dickinson. G. W. Procop has received research funding from of studies to date evaluating MALDI-TOF MS methods for iden- bioMérieux, Bruker, the CDC, and Luminex. S. S. Richter has received tification of yeasts have suggested the use of a more labor-inten- research funding from bioMérieux, Nanosphere, and Forest Laboratories.
sive formic acid/organic solvent extraction method. This method C.-A. D. Burnham has received research funding from bioMérieux, Ac- involves a series of centrifugation steps and is thought to be nec- clerate, Cepheid, and T2 Biosystems. The other authors have no conflicts essary for reliable identification of these organisms, because of the to disclose.
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