EVOLUTION OF THE VITEK MS - bioMérieux Clinical Diagnostics

Transcription

EVOLUTION OF THE VITEK® MS
Axima@SARAMIS™ (Research Use Only)
VITEK MS RUO (Research Use Only)
SARAMIS database
VITEK MS IVD V1 Pre-commercialized database
bioMérieux developed database
VITEK MS IVD V1
VITEK MS IVD V2
VITEK MS Plus (IVD Plus RUO)
VITEK MS IVD V3
Next VITEK MS IVD version release
INTRODUCTION
The VITEK® MS* system is a MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time of Flight)
Mass Spectrometry system that has been designed to provide an accurate and reliable identification result
rapidly to the microbiology laboratory. Moreover, the VITEK MS system aids the physician in diagnosing
and confirming microbial infections quickly which ultimately helps reduce the time for effective treatment
and management of patients with infectious diseases.
MALDI-TOF MS allows for the detection of high-abundance soluble proteins, including ribosomal and other
structural proteins, directly from intact microbial cells resulting in spectra that are analyzed with the
VITEK MS system. The VITEK MS database has been created by collecting mass spectra from multiple
isolates per species, geographically diverse isolates, different sample origins, and different media with different
incubation times.
The VITEK MS system has a comprehensive database of clinically relevant species that allows the
identification of organisms in a matter of minutes. Compared with conventional phenotype or PCR-based
identification, MALDI-TOF MS shows rapid turnaround time (1-2 minutes per sample), low sample
volume requirements, and low reagent costs.
The VITEK MS legacy began with Axima@SARAMIS™, evolving into the VITEK MS RUO system and
then into the VITEK MS IVD system, as shown on the opposite page.
The VITEK MS system includes the VITEK MS Prep Station, VITEK MS Acquisition Station and Myla® software.
The VITEK MS Plus system integrates both the VITEK MS routine database with the open VITEK MS RUO
database (also known as the SARAMIS database)
• The VITEK MS Prep Station securely links specimen information with each spot on the target slide
and to the VITEK 2 Cassette containing the Susceptibility card.
• T he VITEK MS Acquisition station receives the spectra data from the instrument which are then
sent to the Myla software.
• M
yla is connected to both the VITEK MS Prep Station and Acquisition Station allowing for complete
traceability, and holds the compute engine software for identification.
* The VITEK® MS system is not available for diagnostic use in all countries.
Contact your local representative for more information on availability in your country
CONTENTS
➔
ARTICLES
VITEK® MS IVD (In Vitro Diagnostic Applications)
An extraction method of positive blood cultures for direct identification of Candida species by
Vitek MS matrix-assisted laser desorption ionization time of flight mass spectrometry.
4
Lavergne RA, Chauvin P, Valentin A, Fillaux J, Roques-Malecaze C, Arnaud S, Menard S, Magnaval JF, Berry A, Cassaing S, Iriart X.
MEDICAL MYCOLOGY 2013 ; Early Online:1-5
Comparison of two matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass
spectrometry methods and API 20AN for identification of clinically relevant anaerobic bacteria.
5
Jamal WY, Shahin M, Rotimi VO.
JOURNAL OF MEDICAL MICROBIOLOGY 2012 Dec 14 [Epub ahead of print]
Comparison of Vitek MS (MALDI-TOF) to standard routine identification methods:
an advance but no panacea.
6
Harris P, Winney I, Ashhurst-Smith C, O’Brien M, Graves S.
PATHOLOGY 2012 ; 44(6): 583-5
Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for
the identification of clinical Enterococcus isolates.
7
Fang H, Ohlsson AK, Ullberg M, Ozenci V.
EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES 2012 ; 31(11): 3073-7
Routine Identification of Medical Fungi by the New Vitek MS Matrix-Assisted Laser Desorption
Ionization-Time of Flight System with a New Time-Effective Strategy.
8
Iriart X, Lavergne RA, Fillaux J, Valentin A, Magnaval JF, Berry A, Cassaing S.
JOURNAL OF CLINICAL MICROBIOLOGY. 2012 ; 50 (6): 2107-10
Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass
spectrometry system for rapid identification of bacteria in routine clinical microbiology.
9
Dubois D, Grare M, Prere MF, Segonds C, Marty N, Oswald E.
JOURNAL OF CLINICAL MICROBIOLOGY 2012 ; 50(8):2568-76
Comparison of the Microflex LT and Vitek MS systems for Routine Identification of Bacteria
by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.
10
Martiny D, Busson L, Wybo I, El Haj RA, Dediste A, Vandenberg O.
JOURNAL OF CLINICAL MICROBIOLOGY. 2012 ; 50(4):1313-25
VITEK® MS RUO (Research Use Only)
Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization-Time
of Flight Mass Spectrometry Systems for Identification of Nonfermenting Gram-Negative Bacilli
Isolated from Cultures from Cystic Fibrosis Patients.
11
Marko DC, Saffert RT, Cunningham SA, Hyman J, Walsh J, Arbefeville S, Howard W, Pruessner J, Safwat N, Cockerill FR, Bossler AD,
Patel R, Richter SS.
JOURNAL OF CLINICAL MICROBIOLOGY. 2012 ; 50(6): 2034-9
Additional Research Capabilities of the VITEK® MS RUO
Rapid Identification of Bacteria and Yeasts from Positive BacT/ALERT Blood Culture Bottles by
Using a Lysis-Filtration Method and MALDI-TOF Mass Spectrum Analysis with SARAMIS Database.
12
Fothergill A, Kasinathan V, Hyman J, Walsh J, Drake T, Wang YF.
JOURNAL OF CLINICAL MICROBIOLOGY 2013 ; 51(3): 805-9
List of Publications on the Axima@SARAMIS™
13
➔
POSTERS
MSACL - February 9-13, 2013 - San Diego, CA, USA
VITEK® MS IVD V3
Identification of Mycobacteria by VITEK® MS Matrix-Assisted Laser Desorption Ionization –Time of
Flight Mass Spectrometry.
15
Deol P., Girard V., Hyman J., Miller E., Dussoulier R., Mailler S., Schrenzel J., Beni A-M., Ninet Bescher B., Walsh J., Gates A.,
Arsac M., Chatellier S., Dunne W. and Van Nuenen M.
ADDITIONAL RESEARCH CAPABILITIES ON THE VITEK® MS RUO
Matrix-Assisted Laser Desorption Ionization –Time of Flight Mass Spectrometry for rapid antibiotic
resistance detection.
18
Mirande C., Canard I., Perrot N., Welker M., Van Belkum A. and Chatellier S.
MICROBES – September 21-23, 2012 – Sheffield, UK
VITEK® MS IVD V1
Identification of Salmonella enterica spp. enterica using the VITEK® MS MALDI-TOF Mass
Spectrometry System.
20
Identification of Streptococcus pneumoniae and Non-pneumococcal Streptococci of the
Streptococcus mitis Group using the VITEK® MS MALDI-TOF Mass Spectrometry System
21
N. Reading, H.M. Kilgariff, N Ratnaraja
ASM - June 16-19, 2012 - San Francisco, CA, USA
Rapid Identification of Bacteria and Yeasts from Positive Blood Culture Bottles by Using a
Lysis-Filtration Method and MALDI-TOF Mass Spectrum Analysis with SARAMIS Database
22
A. Fothergill, V. Kasinathan, J. Hyman, J. Walsh, T. Drake, X. Huang, E. M. Burd, Y. F. Wang.
ECCMID - March 31–April 3, 2012 – London, UK
VITEK® MS IVD V1
Optimized integration of new technologies (PREVI® Isola and VITEK® MS ) in a
microbiology laboratory using the Lean 6 Sigma methodology
24
Rapid and accurate identification of Campylobacter jejuni and Campylobacter coli isolates using
the VITEK® MS MALDI-TOF mass spectrometry system
26
Evaluation of the VITEK® MS MALDI-TOF mass spectrometry system in a routine clinical laboratory
27
J. Collard, G. Habib J. Djapo Tiani, L. Van Helleputte, H. Palumbo.
N. Reading, A. Dadrah, A. Symonds, H.M. Kilgariff, N. Ratnaraja
Clinical Testing of Bacteria and Yeast from Pediatric Patients by Using MALDI-TOF
VITEK® MS System
V. Kasinathan, X. Zheng, A. Fothergill, D. Carter, Y.F. Wang
28
VITEK® MS IVD
VITEK® MS IVD V1
February, 2013
A specified research extraction protocol was used with the final organism identification performed on the VITEK MS IVD V1 database
MEDICAL MYCOLOGY
2013; Early Online:1-5
An extraction method of positive blood cultures
for direct identification of Candida species by
Vitek MS matrix-assisted laser desorption
ionization time of flight mass spectrometry.
Lavergne RA, Chauvin P, Valentin A, Fillaux J, Roques-Malecaze C, Arnaud S, Menard S, Magnaval JF,
Berry A, Cassaing S, Iriart X.
Complete identification of yeasts with conventional methods currently takes at least 48 hours and sometimes
several days. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)
is a promising alternative allowing faster identification of yeasts and more rapid initiation of antifungal therapy.
This study reports the first evaluation of an extraction method associated with the VITEK® MS matrix-assisted
laser desorption ionization time of flight mass spectrometry for direct identification of Candida species from
positive blood cultures.
This protocol was evaluated with blood cultures that were inoculated with reference and routine isolates (eight
reference strains, 30 patients isolates and six mixed cultures containing two strains of different Candida species),
or from patients with candidemia (28 isolates).
A total of 97% of all isolates included in the study were correctly identified, showing that the extraction protocol used
with the VITEK® MS system performed extremely well with blood cultures of single Candida spp. Nevertheless,
subculture remains indispensable to test fungal resistance and to detect mixed infections. This method significantly
reduced the time of diagnosis and is easily adaptable for use in routine culture.
“This easy extraction method combined with the VITEK® MS system
constitutes a powerful alternative for the identification of Candida spp. directly from
positive blood cultures.”
KEY POINTS
➔ 97% of all isolates included in this study were correctly identified with the use of the study’s extraction protocol.
➔ The VITEK® MS used in conjunction with this extraction method can significantly reduce the time of diagnosis.
4
VITEK® MS IVD
VITEK® MS IVD V1December, 2012
JOURNAL OF MEDICAL MICROBIOLOGY
2012 Dec 14. [Epub ahead of print]
Comparison of two matrix-assisted laser desorption
ionization-time of flight (MALDI-TOF) mass
spectrometry methods and API 20AN for identification
of clinically relevant anaerobic bacteria.
Jamal WY, Shahin M, Rotimi VO.
The study evaluated two commercially available MALDI-TOF MS systems, Bruker Microflex MS and bioMérieux
VITEK® MS, for identification of 274 clinically significant anaerobic bacteria recovered from routine cultures
of clinical specimens in parallel with conventional biochemical (API® 20AN) or molecular methods. Discrepant
results that failed to provide acceptable MALDI-TOF identifications were resolved by gold standard 16S gene
sequencing.
VITEK® MS gave high confidence identification of the 274 isolates, all of which were correctly identified. Bruker
Microflex MS system also gave high confidence identification for 272 of the 274 specimens. After discrepancy
testing, the Bruker MS results agreed with biochemical or molecular method for 89.1% of the isolates at species
level, 10.2% at genus level (0.72% were misidentified). With VITEK® MS, the level of agreement was 100%
species, 100% genus and none were misidentified.
The data provided in this study suggests that implementation of MALDI-TOF MS as a first step for identification
will shorten the turnaround time and reduce costs in the Anaerobe Microbiology Laboratory.
“MALDI-TOF is a rapid, simple, inexpensive technique….
that can easily be implemented in the routine conventional laboratory.”
KEY POINTS
➔ T he VITEK® MS correctly identified ALL 274 clinical anaerobic isolates to the species level compared to 89.1%
by the Bruker Microflex™
➔ The MALDI-TOF MS can reduce reagent use (cost) and labor cost significantly.
5
VITEK® MS IVD
VITEK® MS IVD V1October, 2012
PATHOLOGY
2012; 44(6):583-5
Comparison of Vitek MS (MALDI-TOF) to standard
routine identification methods: an advance but no panacea.
Harris P, Winney I, Ashhurst-Smith C, O’Brien M, Graves S.
A parallel verification trial was conducted between the VITEK MS and routine identifications (VITEK 2, API,
BBL, RapID ANAII, and other rapid bench tests). The study consisted of 750 pure isolates from 150 different
species of bacteria and yeasts. 695 (93%) were wild-type strains obtained from clinical specimens, 39 (5%) were
stored external Quality Assurance Program (QAP) organisms from the Royal College of Pathologists of Australasia
(RCPA) and 16 (2%) were reference strains from the American Type Culture Collection (ATCC).
All isolates were tested with ∂-cyano-4-hydroxycinnamic acid matrix solutions. All suspected yeasts were treated
with 25% formic acid. If the results of the VITEK MS and routine methods were unresolved, then isolates were
referred to a reference laboratory for definitive identification, including 16S rDNA sequencing.
Out of the 750 isolates, 707 (94.3%) showed concordance between the VITEK MS and standard identification
methods to the genus level, and 639 (85.2%) to the species level.
The VITEK MS was found to be reliable and accurate for routine microbial identification in most instances. There
is a significant potential to reduce turnaround times, improve clinical patient care with timely antibiotic therapy,
enhance identification of some challenging organisms, and provide long-term cost savings. In some areas, the
database seems limited. Therefore, future enhancements of the database including additional species is needed.
“The ease and reliability of MALDI-TOF MS allows less reliance
on several additional tests… providing further savings.”
KEY POINTS
➔ VITEK® MS accurately identifies the most common pathogens encountered in routine laboratory work.
➔ VITEK® MS improves identification of some uncommon organisms that may be difficult to identify.
6
VITEK® MS IVD
VITEK® MS IVD V1
June, 2012
EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES
2012;31(11):3073-7
Evaluation of species-specific PCR, Bruker MS,
VITEK MS and the VITEK 2 system for the
identification of clinical Enterococcus isolates
Fang H, Ohlsson AK, Ullberg M, Ozenci V
This study compared the performance of the most relevant diagnostic methods available for the identification of
clinical Enterococcus species, as follows:(1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium,
ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the
groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS;
(3) VITEK® MS; and (4) the VITEK® 2 system. 16S rRNA gene sequencing was used as a reference method in
the study.
The 132 isolates included in the study were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and
21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly
at the species level. The VITEK 2 system could identify 131/132 (99.2 %) and 121/132 (91.7 %) of the isolates at
the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and
81.3 % (13/16) of the E. casseliflavus isolates.
The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF
MS is a rapid, reliable and cost-effective identification technique for enterococci.
“The developments in matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI–TOF) are rapidly changing the routine
diagnostics scene in clinical microbiology laboratories.”
KEY POINTS
➔ PCR assay results are available in hours whereas MALDI-TOF results are available in minutes.
KEY POINTS
➔A
ccurate identification of enterococcal isolates at the species level is important for early appropriate antimicrobial
therapy as well as surveillance.
➔ The results between the PCR assays and MALDI-TOF identified all isolates correctly.
7
VITEK® MS IVD
VITEK® MS IVD V1
June, 2012
JOURNAL OF CLINICAL MICROBIOLOGY
2012;50(6):2107-10.
Routine Identification of Medical Fungi by the
New Vitek MS Matrix-Assisted Laser Desorption
Ionization-Time of Flight System with a New
Time-Effective Strategy.
Iriart X, Lavergne RA, Fillaux J, Valentin A, Magnaval JF, Berry A, Cassaing S.
The study evaluated the VITEK® MS system for rapid fungal identification. A strategy using a single deposit
without prior protein extraction was utilized to save time and money. Clinical isolates were used to evaluate the
performance of the VITEK® MS compared to that of both routine laboratory techniques and VITEK® 2.
236 isolates, representing 27 species of fungi, were analyzed. With all species included, the VITEK® MS performed
well in the identification of yeasts and Aspergillus fungi (93.2% of correct identifications). Considering only the
species present in the VITEK® MS database, 98.2% of the total yeasts had a correct identification and 100% of
Aspergillus species.
The VITEK® MS system also represents a major improvement in terms of time and money saving.
“The new VITEK® MS system …
has an excellent performance profile for the identification of a large panel
of yeasts and for Aspergillus fungi”
KEY POINTS
➔A
ll of the Aspergillus isolates included in the database were correctly identified by VITEK® MS when compared to DNA
sequencing results.
➔
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utilizing
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®
The VITEK MS used in conjunction with this extraction method can significantly reduce the time of diagnosis.
8
VITEK® MS IVD
VITEK® MS IVD V1
May, 2012
2012;50(8):2568-76
Performances of the Vitek MS matrix-assisted laser
desorption ionization-time of flight mass
spectrometry system for rapid identification of
bacteria in routine clinical microbiology.
Dubois D, Grare M, Prere MF, Segonds C, Marty N, Oswald E.
The study objective was to assess the performance and technical practicability of the VITEK® MS system, using a
single deposit and without prior extraction step from bacterial colonies.
767 routine clinical isolates representative of 50 genera and 124 species were tested on the VITEK® MS and then
compared with reference identifications obtained mainly with the VITEK® 2 phenotypic system. If identifications
were discordant, molecular techniques provided reference identifications. Using an original spectra classifier
algorithm, the VITEK® MS system provided 96.2% correct identifications: to the species level (86.7%), to the
genus level (8.2%), or within a range of species belonging to different genera (1.3%).
1.3% of isolates were misidentified and 2.5% were unidentified, partly because the species was not included in
the database. A second deposit provided a successful identification for 0.8% of isolates unidentified with the first
deposit.
The VITEK® MS system is a simple, convenient, and accurate method for fast bacterial identification with a single
deposit and without any extraction step. In addition to a second deposit in uncommon cases, expanding the spectral
database is expected to further enhance performance. Technical ownership of the VITEK® MS is straightforward
and fast,
“The VITEK® MS system allows with only one deposit of crude bacteria
and without any extraction step, a fast and reliable acquisition of bacterial ID
for most bacterial species isolated routinely in a medical laboratory.”
KEY POINTS
➔ T he VITEK® MS system generated a low frequency of unusable spectra without the use of a formic acid-based protein
extraction.
➔ The VITEK® MS is able to discriminate S. pneumonia, a pathogen species from other alpha-hemolytic streptococci.
9
VITEK® MS IVD
VITEK® MS IVD V1 PRE-COMMERCIALIZED DATABASE
April, 2012
2012;50(4):1313-25.
Comparison of the Microflex LT and Vitek MS
systems for Routine Identification of Bacteria by
Matrix-Assisted Laser Desorption Ionization-Time of
Flight Mass Spectrometry.
Martiny D, Busson L, Wybo I, El Haj RA, Dediste A, Vandenberg O.
The study compared the performance of MALDI-TOF systems currently commercialized in Europe regarding
analytical accuracy and practicability to determine the best choice for use in a routine bacteriology laboratory.
1,129 isolates, including 1,003 routine isolates, 73 anaerobes, and 53 bacterial enteropathogens, were tested on the
Microflex LT and Axima Assurance devices. The spectra were analyzed using three databases: Biotyper (Bruker
Daltonics), Saramis, and VITEK® MS (bioMérieux). Among the routine isolates requiring identification to the
species level (n = 986), 92.7% and 93.2% were correctly identified by the Biotyper and VITEK® MS databases,
respectively. The VITEK® MS database is more specific for the identification of Streptococcus viridans. For the
anaerobes, the Biotyper database often identified Fusobacterium isolates to only the genus level, which is of low
clinical significance, whereas 20% of the Bacteroides species were not identified or were misidentified by the
VITEK® MS database. For the enteropathogens, the poor discrimination between Escherichia coli and Shigella
explains the high proportion of unidentified organisms. In contrast to the Biotyper database, the VITEK® MS
database correctly discriminated all of the Salmonella enterica serovar Typhi isolates (n = 5).
This study demonstrated the Microflex LT and VITEK® MS systems to be equally good choices in terms of
analytical efficiency for routine procedures. Other factors including price, work flow, and lab activity, will affect
the individual laboratory’s choice of a system.
“The VITEK® MS IVD system will likely
simplify laboratory quality management”
KEY POINTS
➔ The VITEK® MS IVD system seemed to have more user-friendly software.
➔ T he VITEK® MS IVD system has a more highly developed quality management system because it contains dedicated
for quality
controls
andstudy
a well-defined
traceability
system.
97%positions
of all isolates
included
in this
were correctly
identified
with the use of the study’s extraction protocol.
® MS used in conjunction with this extraction method can significantly reduce the time of diagnosis.
The
VITEK
➔ The
accuracy
of the Biotyper database was found to be lower than that of the Saramis™ and VITEK® MS database
10
VITEK® MS RUO
VITEK® MS RUOJune, 2012
2012;50(6):2034-9.
Evaluation of the Bruker Biotyper and Vitek MS
Matrix-Assisted Laser Desorption Ionization-Time of
Flight Mass Spectrometry Systems for Identification
of Nonfermenting Gram-Negative Bacilli Isolated from
Cultures from Cystic Fibrosis Patients.
Marko DC, Saffert RT, Cunningham SA, Hyman J, Walsh J, Arbefeville S, Howard W, Pruessner J, Safwat N, Cockerill FR,
Bossler AD, Patel R, Richter SS.
This blinded study evaluated Bruker Biotyper and VITEK® MS MALDI-TOF systems for the identification of
non-fermenting Gram-negative bacilli (NFGNB) isolated from cystic fibrosis cultures compared to conventional
biochemical or molecular methods.
Two hundred NFGNB recovered from respiratory cultures from cystic fibrosis patients were sent to Mayo Clinic
for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with VITEK® MS
(SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDITOF identification, an extraction procedure was performed.
The MS identifications from both of these systems were compared to the biochemical or molecular identification
that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene
sequencing at the University of Iowa Health Care (UIHC).
After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method with 72.5%
of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level
of agreement for VITEK® MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified).
Both MS systems provided rapid (≤3 min per isolate) and reliable identifications.
The identification of NFGNB by both MALDI-TOF MS systems was superior to conventional biochemical
methods. The agreement of combined species/complex/genus-level identification with the reference method
was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often.
Species-level agreement with the reference method was similar for both MS systems (72.5% and 80% respectively,
P = 0.099).
“The accuracy, ease of use, low reagent cost, and speed of MALDI-TOF MS
support the implementation of this technology for identification of NFGNB”
KEY POINTS
➔ T he final identifications of P. aeruginosa, the predominant NFGNB isolated from CF patients, were 100% concordant
KEY POINTS
with biochemical or molecular methods.
➔W
hen looking at all 16 B. cepacia complex isolates, the VITEK® MS RUO provided more species level identifications that
agreed with the reference method than the Bruker system, without needing any prior extraction.
11
Additional Research Capabilities of the VITEK® MS RUO
VITEK® MS RUO
December, 2012
2013;51(3):805-9
Rapid Identification of Bacteria and Yeasts from Positive
BacT/ALERT Blood Culture Bottles by Using a
Lysis-Filtration Method and MALDI-TOF Mass Spectrum
Analysis with SARAMIS Database.
Fothergill A, Kasinathan V, Hyman J, Walsh J, Drake T, Wang YF
This study evaluates the performance of a novel filtration-based method for processing positive BacT/ALERT®
blood culture broth for immediate identification of microorganisms by MALDI-TOF VITEK® MS RUO (VMS).
BacT/ALERT® non-charcoal based blood culture bottles that were flagged as positive by the BacT/ALERT®3D
system were included. An aliquot of positive blood culture broth was incubated with lysis buffer for 2-4 minutes
at room temperature, the resulting lysate was filtered through a membrane, and harvested microorganisms were
identified by VMS. The lysis buffer used in this protocol eliminates blood cells, but leaves microorganisms intact
for rapid analysis by MALDI-TOF MS.
A total of 259 bottles were included in the study, comprising 225 monomicrobic and 28 polymicrobic positive
blood cultures (6 bottles were negative on subculture). The VMS identified 189 (73%) cultures to the species level,
51 (19.7%) gave no identification (ID), while 6 (2.3%) gave identifications that were considered incorrect. Among
131 monomicrobic isolates from positive blood bottles with one spot having a score of 99.9%, all were correctly
identified to the species level (100%). In 202 bottles where VMS was able to generate an ID, 189 (93.6%) were
correct to the species level, whereas the IDs provided for 7 isolates (3.5%) were incorrect.
In conclusion, this method does not require centrifugation and produces a clean spectrum for VMS analysis in
less than 15 minutes. This study demonstrates the effectiveness of the new Lysis-Filtration method for identifying
microorganisms directly from positive blood culture bottles in a clinical setting.
“… the VMS when used in combination with the ‘direct-from-positive blood culture’ method
described herein has the potential to greatly reduce the time to identification
of possible agents of bacteremia/sepsis and to improve the delivery of appropriate
antimicrobial therapy to the patient”
KEY POINTS
➔ The lysis buffer used in this protocol eliminates blood cells while leaving microorganisms intact.
12
➔ T his method does not require centrifugation and produces a clean, concentrated sample of microorganism in less than
15 minutes.
Research Use Only
List of Publications on the Axima@SARAMIS™
Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods
for the identification of clinically relevant anaerobic bacteria.
Veloo AC, Knoester M, Degener JE, Kuijper EJ.
CLINICAL MICROBIOLOGY AND INFECTION 2011;17(10):1501-6.
Improved clinical laboratory identification of human pathogenic yeasts by matrix-assisted laser desorption
ionization time-of-flight mass spectrometry.
Bader O, Weig M, Taverne-Ghadwal L, Lugert R, Gross U, Kuhns M.
CLINICAL MICROBIOLOGY AND INFECTION 2011;17(9):1359-65.
Recognition of Clostridium difficile PCR-ribotypes 001, 027 and 126/078 using an extended
MALDI-TOF MS system.
Reil M, Erhard M, Kuijper EJ, Kist M, Zaiss H, Witte W, Gruber H, Borgmann S.
EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES 2011;30(11):1431-6.
Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap
gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Bergeron M, Dauwalder O, Gouy M, Freydiere AM, Bes M, Meugnier H, Benito Y, Etienne J, Lina G, Vandenesch F, Boisset S.
EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES 2011;30(3):343-54.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically
relevant bacteria.
Benagli C, Rossi V, Dolina M, Tonolla M, Petrini O.
PLoS One. 2011 25;6(1):e16424.
Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting.
Gaia V, Casati S, Tonolla M.
SYSTEMATIC AND APPLIED MICROBIOLOGY 2011;34(1):40-44.
Identification of Staphylococcus intermedius Group by MALDI-TOF MS.
Decristophoris P, Fasola A, Benagli C, Tonolla M, Petrini O.
Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry
in comparison to sodA sequence analyses.
Hinse D, Vollmer T, Erhard M, Welker M, Moore ER, Kleesiek K, Dreier J.
Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry.
Veloo AC, Erhard M, Welker M, Welling GW, Degener JE
13
Research Use Only
List of Publications on the Axima@SARAMIS™ (continued)
Rapid genus- and species-specific identification of Cronobacter spp. by matrix-assisted laser desorption
ionization-time of flight mass spectrometry.
Stephan R, Ziegler D, Pflüger V, Vogel G, Lehner A.
JOURNAL OF CLINICAL MICROBIOLOGY 2010;48(8):2846-51
Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with
conventional phenotypic identification for routine identification of bacteria to the species level.
Cherkaoui A, Hibbs J, Emonet S, Tangomo M, Girard M, Francois P, Schrenzel J.
JOURNAL OF CLINICAL MICROBIOLOGY 2010;48(4):1169-75.
Identification of dermatophyte species causing onychomycosis and tinea pedis by
MALDI-TOF mass spectrometry.
Erhard M, Hipler UC, Burmester A, Brakhage AA, Wöstemeyer J.
EXPERIMENTAL DERMATOLOGY 2008;17(4):356-61.
14
➔ MSACL / February, 2013
San Diego, CA, USA
VITEK® MS IVD V3
Identification of Mycobacteria by VITEK® MS MatrixAssisted Laser Desorption Ionization –Time of Flight Mass
Spectrometry
Deol P.1, Girard V. 2, Hyman J.1, Miller E.1, Dussoulier R.1, Mailler S.2, Schrenzel J.3, Beni A-M.3, Ninet Bescher B.3, Walsh
J.1, Gates A.1, Arsac M.2, Chatellier S.2, Dunne W.1 and Van Nuenen M.1
1/bioMérieux Inc., 100 Rodolphe St, Durham, NC, 27712 - 2/bioMérieux, Route de Port Michaud, 38390 La Balme Les Grottes, France
3/Hôpitaux Universitaires de Genève, Laboratoire de Bactériologie, Rue Gabrielle Perret Gentil 4, 1211 Geneve 14, Switzerland
ABSTRACT
VITEK MS* consists of a MALDI-TOF mass spectrometer for microbial
identification and a database recognizing unique fingerprints.
®
Figure 1: Fluorescent staining results with bead beating, horizontal vortexing, and
without mechanical disruption
Mycobacterial species require inactivation and protein extraction
due to their pathogenicity and lipid rich cell wall.
The study describes a sample preparation method using mechanical disruption and chemical treatment tested f rom multiple growth
media for spectral database development. Most of the 37 mycobacterial species, tested with an average of 8 strains per species,
were clearly differentiated except the M. tuberculosis complex and
M.fortuitum/M.porcinum.
The identification results obtained using this sample preparation
method and the mycobacteria database indicated a 100% species
match for 34 species and 94-98% for the remaining 3 species.
The VITEK® MS platform offers rapid, reliable, and robust microbial
identification with high throughput.
INTRODUCTION
An accurate identification of mycobacteria species is essential for
epidemiology, proper diagnosis, and administration of appropriate
antimicrobial therapy.
Identification of mycobacteria species represents a technical challenge
for sample preparation due to cellular structure and pathogenicity.
The processing procedure must ensure that organisms are rendered
nonviable for safe handling outside of a biosafety level-3 environment
and that both hydrophobic and hydrophilic cellular proteins are made
accessible for subsequent ionization.
This study describes a mycobacterial sample preparation method for
use with VITEK® MS that ensures organism inactivation and extraction
of proteins from cultures grown on solid (LJ and 7H11 agar) and in
liquid (BacT/ALERT® MP and BACTEC™ MGIT™ 960) media. The
inactivation and extraction method is suitable for routine laboratory
work where simplification of workflow and safety are key factors.
Yellowish green live cells
on red background
Red background with cell debris
MATERIALS AND METHODS
Drug-susceptible and resistant strains of M. tuberculosis and nontuberculous mycobacteria (NTM) were used for the inactivation study.
Optimal inactivation is achieved using mechanical disruption (bead
beating for 5 min or vortexing for 15 min) with 0.5 mm glass beads in
a pre-sterilized vial containing 70% ethanol (Figure 1).
After disruption, the sample is incubated for 10 min at room temperature in the same tube, and then centrifuged. The pellet is reconstituted
with formic acid then acetonitrile is added. After centrifugation, the
supernatant is placed on the target slide and overlain CHCA matrix.
Figure 2 shows the steps of the sample processing method.
For protein extraction, 2-20 strains per species (an average of 8
strains) of mycobacteria were grown on solid (LJ/Coletsos and 7H11
agar) and in liquid media (BacT/ALERT® MP and BACTEC™ MGIT™
960).
For the purpose of database development, all spectra were checked
visually for quality and those with 80-200 peaks were considered.
Cluster analysis was performed for each species of mycobacteria to
assess intra-species diversity. DNA sequencing (16S or rpoB) was
performed on all outlier strains for confirmation or inclusion/exclusion
of spectra.
Multidimensional analysis was conducted to evaluate the differentiation
of species (Figure 3 is an example with a subset of the 37 species).
VITEK® MS has not been cleared by the United States FDA for sale in the USA and is therefore not yet commercially available in the USA.
15
Identification of Mycobacteria by VITEK® MS Matrix-Assisted Laser Desorption Ionization
-Time of Flight Mass Spectrometry
Figure 2: Sample processing method from solid and liquid media.
Growth from solid medium
Centrifuge and discard
supernatant
Resuspend
1 ul loopful of growth
in 500 ul 70% EtOH
(in vial containing
0.5 mm glass beads)
Resuspend in 500 ul
70% EtOH and transfert
suspension
(to vial containing 0.5 mm glass beads)
Growth from liquid medium
Take aliquot
(2 ml from MGIT/
3 ml from MP Bottle)
+MGIT / MIP bottle
Mycobacteria Sample
If bead beater
not available,
Vortex for 15 min,
incubate 10 min **
«Bead Beat» for 5 min, incubate
10 minutes (inactivation time)
Vortex, transfert suspension
to empty vial
E.coli ATCC 8739
18-24 hours old
Apply 1 ul
loopful directly
to target slide
Inoculate 1 ul
CHCA Matrix
on target slide
Centrifuge (e.g. 10,000 rpm for 2 min),
remove EtOH supernatant
Add 10 ul 70% formic acid, vortex
Control Sample
Add 10 ul acetonitrile,
centrifuge
Inoculate 1 ul of suspension
on target slide
Test in VITEK MS
RESULTS
CONCLUSION
The samples with mechanical disruption either by bead beater or vortex
at recommended times clearly show cell disruption as compared to
the control samples without disruption (Figure 1). No growth was
observed in all media types tested for 42 days.
Mechanical disruption followed by 70% ethanol treatment is a quick
and effective inactivation method for mycobacteria species. Extraction
of proteins is performed in a minimal volume to achieve good quality
spectra. The method is safe, short, requires less manipulation and is
suitable for routine laboratory work.
The process from inactivation of mycobacteria to slide preparation
takes 20-35 minutes depending upon number of samples and mechanical
disruption method. The extracted proteins are concentrated in a minimal
final volume. The process is standardized for cultures grown on solid
and in liquid media.
The mycobacterial database is currently comprised of 1286 spectra.
Among the M. tuberculosis complex species tested, M. tuberculosis,
M. bovis, and M. africanum spectra look very similar and the same
finding was observed for M. fortuitum and M. porcinum (as shown in
the multidimensional analysis in Figure 3).
Clinically relevant NTM such as
M. scrofulaceum, M. kansasii,
M. haemophilum, and M. xenopi
The rapid growing mycobacteria
M. chelonae, and M. smegmatis
patterns (Figure 4).
M. avium, M. intracellulare,
M. gordonae, M. marinum,
indicated clear differentiation.
M. abscessus, M. fortuitum,
also exhibited distinguishable
Cluster analysis based on peak similarity in the spectra indicated
consistent results with different media for each mycobacterial species
tested. Figure 5 shows the similarity of spectra of M. tuberculosis
25177 grown in different media types.
Preliminary identification results demonstrated 100% species match
for 34 mycobacterial species and 94-98% match for the remaining 3.
16
The VITEK® MS Mycobacteria database currently consists
of 1286 spectra from 37 species (2-20 strains per species).
Preliminary identification results demonstrated 100% match
for 34 species and 94-98% match for the remaining 3.
Work is in progress to enhance the VITEK® MS database with additional
mycobacteria species.
Identification of Mycobacteria by VITEK® MS Matrix-Assisted Laser Desorption Ionization
-Time of Flight Mass Spectrometry
Figure 3: Multidimensional analysis showing distinction and overlaps between Mycobacterial species
MDS colored according to Mycobacteria species
Kansasii
Fortuitum,
Porcinum
Africanum, Bovis,
Tuberculosis
Avium
Genavense
Figure 4: Cluster analysis showing distinction between rapid growing mycobacterial species
M. abscessus, M. chelonae, M. smegmatis and M. fortuitum
M. abscessus
M. chelonae
M. smegmatis
M. fortuitum
Figure 5: Spectra of M. tuberculosis 25177 from different media types.
17
➔ MSACL / February, 2013
San Diego, CA, USA
Additional Research Capabilities on the VITEK® MS RUO
Matrix-Assisted Laser Desorption Ionization –Time of Flight
Mass Spectrometry for rapid antibiotic resistance detection
Mirande C., Canard I., Perrot N., Welker M., Van Belkum A. and Chatellier S.
Research & Development Microbiology, bioMérieux, La Balme-les-Grottes, France
ABSTRACT
Hydrolytic enzymes play a major role in antibiotic resistance of
Gram negative bacterial species. These enzymes hydrolyze certain
antibiotics, leading to their inactivation. Our work shows that
Matrix Assisted Laser Desorption Ionization-Time of Flight Mass
Spectrometry (MALDI-ToF MS) is a rapid and well-adapted tool for
the analysis of antibiotic degradation. Herein, we describe a fast
assay to monitor faropenem hydrolysis by resistant bacteria.
Carbapenem-sensitive strains were used as β-lactamase negative
controls. Well characterized K. pneumoniae isolates producing
carbapenemases were included in the comparative hydrolysis assays
(Table 2). Susceptibility results and resistance genes for each strain were
determined by VITEK®2 antimicrobial susceptibility cards (bioMérieux,
Marcy l’Etoile, France) and by PCR.
A commercially available faropenem solution was used in combination
with the different strains. Bacteria were re-suspended (0.5, 1 or 2
McF) in the antibiotic solution (0.1, 0.25 and 0.5 mg/mL in H2O) and
incubated at 37°C for 3 hours. Subsequently, the tubes were centrifuged
for 2 min at 13,000g at room temperature (Figure 2).
For the first time, the bioMérieux VITEK® MS RUO instrument was
used for such an approach and this instrument was shown to be
suitable for qualitative measurements of enzyme-mediated drug
degradation.
INTRODUCTION
Matrix-assisted laser desorption ionization–time of flight (MALDI-ToF)
mass spectrometry (MS) has been introduced into routine microbiological laboratories for the identification of bacteria and fungi and
may also be applied in other aspects of the global clinical diagnostic
process.
A common resistance mechanism developed or acquired by different
bacterial species involves the inactivation of β-lactam antibiotics by the
expression of enzymes which hydrolyze the β- lactam ring (Figure 1).
Figure 1: Native and hydrolysed form of faropenem
HO
H
HO
H
H
S
ß-lactamase
NH
H
O
O
O
Faropenem
Surpernatant
A solution to this issue may involve using a MALDI-ToF MS to detect
metabolites produced after β-lactamase hydrolysis rather than the
β-lactamase itself. In mass spectra, hydrolysis of β-lactam ring results
in disappearance of the original mass peak through a molecular mass
shift of +18 Da (Table 1).
Here we describe a rapid method for detection of the β-lactam faropenem
hydrolysis and direct detection of carbapenemase activity with bioMérieux
VITEK® MS RUO instrument.
Table 1: Calculated masses (Da) and corresponding molecular structures of faropenem
Native forms
VITEK®MS RUO
OH
Inactive Faropenem (hydrolysed)
Today, the rapid detection of carbapenemases still is a major challenge
in microbiological diagnostics. Direct detection of enzymes has remained
elusive because many proteins involved in drug resistance are frequently
not expressed at high levels compared to other bacterial proteins.
Hydrolysed forms (+18 Da)
M+H
M+Na
M+2Na
M+3Na
M+H
M+Na
M+2Na
M+3Na
286,3
308,3
330,3
352,3
304,3
326,3
348,3
370,3
18
37°C - 3h
Mass
spectra
analysis
H
OH
OH
O
Faropenem
O
O
N
Figure 2: Methodology for antibiotic resistant detection
Picking
colonies
H
S
The cell-free supernatant was analyzed by bioMérieux VITEK® MS RUO
instrument in the 200-600 m/z mass range. Because background
signals may confound these spectra, we tested α-cyano-4hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB)
as matrix at various concentrations and diluted in ethanol, acetonitrile
and/or water (in different proportions).
RESULTS
Optimization of hydrolysis detection : Spectra without background
peak in the interval of 280-400 m/z were obtained using 10 mg/mL
of CHCA in ethanol, acetonitrile and water (30/30/30). Optimal
detection was obtained with the ratio antibiotic – bacterial inoculum :
0.25mg/mL - 2McF, respectively.
Carbapenem-sensitive strains
Spectra obtained after incubation with faropenem revealed the
molecular peak of sodium salts of faropenem [M+Na]+ at m/z
308.3 and [M+2Na]+ at m/z 330.3.
Carbapenemase-producing strains
Spectra revealed decreased intensities of the peaks at m/z 308.3
and 330.3. Additional peaks at m/z 304.3, 326.3 and 348.3
appeared ➔ Hydrolyzed forms of faropenem (Table 1 and
Figure 3).
Matrix-Assisted Laser desorption Ionization -Time of Flight Mass Spectrometry for rapid
antibiotic resistance detection
Table 2: Bacterial strains characteristics and VITEK®MS RUO results
Ratio of peak intensities at 308/379 was
calculated and CHCA (m/z 379) was used as an
internal standard (Table 2).
MALDI-ToF MS
Analysis
Bacterial strains
Isolate ref
Resistance mechanism*
Result
Ratio 308/379
T0
T3h
K. pneumoniae
1108001
KPC2
Degradation
0.45
0.03
K. pneumoniae
0601009
KPC
Degradation
0.46
0.34
K. pneumoniae
0505049
KPC
Degradation
0.56
0.43
K. pneumoniae
0601007
KPC
Degradation
0.58
0.48
K. pneumoniae
11085008
KPC2
Degradation
0.70
0.57
K. pneumoniae
1108003
KPC3
Degradation
0.43
0.36
K. pneumoniae
1108002
KPC3
Degradation
0.51
0.34
K. pneumoniae
0505034
KPC
No Degradation
0.38
0.49
K. pneumoniae
0603004
KPC
No Degradation
0.38
0.38
E.coli
0607051
ESBL
No Degradation
0.75
0.77
P. mirabilis
0607045
ESBL
No Degradation
0.79
0.81
E.coli
0804151
HL Case
No Degradation
0.17
0.22
M. morganii
0504063
HL Case
No Degradation
0.29
0.38
S. aureus
7509008
/
No Degradation
0.39
0.40
K. pneumoniae
1002015
/
No Degradation
0.82
0.88
E.coli
7308009
/
No Degradation
0.36
0.37
Negative control
/
/
No Degradation
0.19
0.21
Ratios clearly decreased for resistant strains except
for two strains n°0505034 and 0603004.
Interestingly, strain n° 1108001 showed an important
ratio decrease compared to the other strains.
Such observation could be explained by the variations of expression at transcriptomic or post-transcriptomic level.
This hypothesis could be verified by molecular
approach.
Rather, negative control (without strain) and
non-carbapenemases-producing strains (without
resistance mechanism, ESBL or HL Case) show a
constant trend between T0 and T3h.
Figure 3: Mass spectra obtained after faropenem hydrolysis assay
CONCLUSION
The MALDI-ToF MS-based analysis of resistance/susceptibility against
β-lactam antibiotics is an approach which has recently been applied
for the analysis of the hydrolysis of ampicillin, ertapenem, and
meropenem by different bacterial strains.
However, no study has yet been carried out with faropenem and with
the bioMérieux VITEK® MS RUO instrument.
VITEK® MS RUO instrument was shown to be suitable for
qualitative measurements of enzyme-mediated drug
degradation.
Future studies will include various antibiotic families and
additional susceptible and resistant strains to expand on this
potentially clinically useful technique.
19
➔ MICROBE / September, 2012
Sheffield (UK) VITEK® MS IVD V1
Identification of Salmonella enterica ssp enterica using
the VITEK® MS MALDI-TOF mass spectrometry system.
N. Reading, H.M. Kilgariff, N. Ratnaraja
Department of Microbiology, Sandwell & West Birmingham Hospitals NHS Trust, Birmingham, United Kingdom.
OBJECTIVES
The VITEK®MS (bioMérieux, France) is a recently launched MALDI-TOF
MS system for rapid identification of bacterial and fungal isolates.
Identification of Salmonella enterica ssp enterica within our laboratory
has previously been carried out using biochemical and serological
methods, with identification often being available 18-24 hours later.
1. Pick colony
2. Add to target slide
3. Add Matrix
Our primary objective was to evaluate the performance of the VITEK® MS
for the identification of Salmonella species including Salmonella typhi
and Salmonella paratyphi.
A second objective was to evaluate the identification achieved when
isolates were tested direct from the selective medium, Xylose Lysine
Desoxycholate Agar (X.L.D.)
METHODS
A total of 89 previously isolated, well characterised strains belonging to
the Salmonella enterica ssp enterica group were tested on the VITEK® MS
system.
This comprised a total of 28 Salmonella typhi strains,19 strains of
Salmonella paratyphi A. with the remaining strains made up of various
Salmonella ssp.
All strains had previously been identified using biochemical and
serological methods. All isolates had been submitted for reference
laboratory confirmation at HPA Centre for Infections, Colindale, UK.
4. Place in VITEK MS
5. Review results
RESULTS
Table of results
Species
Salmonella
typhi Total Species Family Incorrect
N° IDID
StrainscorrectGroup identificationidentificationspecies group
n=83correct
28 27 1
0
0
99.3% 100%
Salmonella
paratyphi A 19 14 5
0
0
73.7% 100%
Salmonella
species 40 1
0
1
95.2% 97.6%
42 35 (100%) isolates of Salmonella species tested directly for X.L.D. agar
correctly identified as Salmonella Group.
The previously isolated strains were cultured onto Columbia Horse
Blood Agar (bioMérieux, France) prior to testing and incubated in air
at 35oC for 24 hours.
When combined, 123/124 (99.2%) isolates correctly identified to
Salmonella group using the VITEK® MS.
A further 35 strains of Salmonella ssp. were isolated on X.L.D. Agar
(bioMérieux, France) as part of routine faeces processing and were
tested direct from X.L.D. media on the VITEK® MS. These strains were
referred to a reference laboratory for confirmation and typing.
The identification of Salmonella species obtained using the VITEK® MS
MALDI-TOF system was accurate when strains were tested directly from
selective media, with 100% of isolates giving the correct identification.
This provides considerable time savings compared to traditional
methods.
Disposable target slides (bioMérieux, France) were inoculated with a
small amount of the test isolate to provide a thin layer of organism1
and overlaid with 1 µL of 𝛼-Cyano-4-hydroxy-cinnamic acid (C.H.C.A.)
matrix solution (bioMérieux, France) and air dried1.
The resulting slides were then processed in the VITEK® MS instrument
with automatic database analysis of the obtained mass spectra within
MYLA® software2 (bioMérieux, France). A second target spot was
analysed if no spectra or identification was obtained.
CONCLUSIONS
Although the system failed to differentiate some organisms within the
Salmonella Group, the given group identification proved to be correct
and complementary serological methods should still be used to
delineate Salmonella strains further.
The VITEK® MS is a fast and reliable method that could
replace traditional biochemical identification methods in
routine clinical laboratories for the identification of Salmonella
species.
REFERENCES:
1. VITEK® MS User Manual, bioMérieux France 2011
2. MYLA® User Manual, bioMérieux France 2011
20
➔ MICROBE / September, 2012
Sheffield (UK) VITEK® MS IVD V1
Identification of Streptococcus pneumoniae and Nonpneumococcal Streptococci of the Streptococcus mitis Group
using the VITEK®MS MALDI-TOF Mass Spectrometry System.
N. Reading, H.M. Kilgariff, N. Ratnaraja
Department of Microbiology, Sandwell & West Birmingham Hospitals NHS Trust, Birmingham United Kingdom.
OBJECTIVES
RESULTS
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI-TOF MS) is a rapid method for identification of
microorganisms.
Table of results
The VITEK®MS (bioMérieux, France) is a recently launched MALDI-TOF
mass spectrometry system for rapid identification of bacterial and
fungal isolates.
The identification of Streptococcus mitis Group members and
differentiation of S. pneumoniae from other group members can be
difficult using traditional methods and even when 16S Sequence based
testing are considered3. Previous studies have shown mass spectrometry
also had varying performance for the analysis of S. mitis Group
streptococci4.
Our objective was to evaluate the performance of the VITEK® MS
in a routine clinical microbiology laboratory for the identification of
Streptococcus pneumoniae and Non-pneumococcal streptococci
belonging to the Streptococcus mitis Group.
Total SpeciesFamily Incorrect
N° IDID
StrainscorrectGroup identificationidentificationspeciesgroup
n=226correct
S. pneumoniae 141 140 0
0
1
99.29% 9 9.29%
S. oralis 44 0
43 1
0
0% 97.73%
S. mitis 31 0
30 1
0
S. pseudopneumoniae
6
6
0
0
0
100% 100%
S. cristatus 4
4
0
0
0
100% 100%
0% 96.77%
Only 1 mucoid isolate of S.pneumoniae failed to identify even on
repeated testing, with 99.29% of isolates being correctly identified by
the VITEK® MS.
1 strain each of S.oralis and S.mitis misidentified as S.pneumoniae,
further testing of each isolate showed the original identification to
be correct.
CONCLUSIONS
METHODS
226 previously identified, well characterised strains belonging to the
Streptococcus mitis Group (141 S. pneumoniae, 44 S. oralis , 31 S. mitis,
6 S. pseudopneumoniae and 4 S. cristatus), isolated between 2004 and
2012, were subcultured onto Columbia Horse Blood Agar (bioMérieux,
France). Strains were grown at 37°C in 5% CO2 for 24 hours.
small amount of a single bacterial colony to provide a thin layer of
organism using a disposable plastic loop.
These were overlaid with 1µL of α-Cyano-4-hydroxycinnamic acid
(C.H.C.A) matrix solution (bioMérieux, France) and allowed to air dry.1
The resulting slides were then analysed in the VITEK® MS instrument,
using the automatic database analysis of the obtained mass spectra
within MYLA® software (bioMérieux, France) to provide isolate
identification.2
A second target spot was analysed if no spectra or no identification
was obtained.
Discordant results were further analysed using susceptibility to EthylHydrocupreine (Optochin) (Thermo-Fisher Scientific, Basingstoke)
and Bile Solubility using 10% Desoxycholate (Thermo-Fisher
Scientific, Basingstoke) solution alongside the VITEK® 2 GP-ID Card
(bioMérieux, France).
1. Pick colony
Species
3. Add Matrix
The VITEK® MS MALDI-TOF mass spectrometry system is a fast,
reliable method to identify Streptococcus mitis Group streptococci
with 98.67% of strains correctly identified to family/group level.
Although the system failed to differentiate S. mitis and S.oralis, the
given group identification or slash-line identification proved to be
correct in the majority of organisms and alternative molecular
methods often struggle to delineate these strains.3,4
Identification was available very rapidly, saving 24 hours
in many cases when compared to traditional phenotypic or
biochemical identification methods.
REFERENCES:
1. VITEK® MS User Manual, bioMérieux, France 2011
2. MYLA® User Manual, bioMérieux, France 2011
3. Richard Facklam, Whatever Happened to the Streptococci. Clin. Microbiol. Rev. 2002,
15(4):613.
4. Christopher D. Doern et al. It’s not easy being green: The Viridans Group Streptococci
J.Clin.Microbiol.2010,48(11):3829
5. Review results
21
➔ ASM / June, 2012
San Francisco, USA Additional Research Capabilities on the VITEK® MS RUO
Rapid Identification of Bacteria and Yeasts from Positive
Blood Culture Bottles by Using a Lysis-Filtration Method and
MALDI-TOF Mass Spectrum Analysis with SARAMIS Database.
A. Fothergill1, V. Kasinathan1, J. Hyman2, J. Walsh2, T. Drake3, X. Huang3, E. M. Burd1, Y. F. Wang1,3;
Emory University School of Medicine, Atlanta, GA, 2bioMérieux, Inc., Durham, NC, 3Grady Memorial Hospital, Atlanta, GA.
1
INTRODUCTION
Rapid identification of bloodstream infections after a positive blood
culture result would greatly improve patient care. Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS) can be used to identify (ID) microorganisms. The VITEK® MS
RUO System with SARAMIS™ database by bioMérieux is a research use
only MALDI-TOF MS system for rapid detection of bacterial and yeast
isolates. MALDI-TOF mass spectrometry (MS) has the potential to serve
as a fast and reliable method for identifying microorganisms.
This study aims to evaluate the performance of a novel filtration-based
method4 for processing positive BacT/ALERT® blood culture broth for
immediate identification by MALDI-TOF MS.
1 μL of CHCA matrix. When a blood culture bottle was repeated,
the volume of blood culture broth and corresponding buffers was
doubled. All other procedures remained the same.
SAMPLE PREPARATION
Figure 1: Sample Preparation
BacT/ALERT® anaerobic (SN) and standard aerobic (SA) noncharcoal
based blood culture bottles that were flagged as positive by
bioMérieux’s BacT/ALERT®3D system were included in the study.
If possible, the bottles were processed the same day they flagged
as positive, but older bottles were included as well. A bottle was
considered to have a valid MALDI result if at least one spot gave
a SARAMIS rating of 75% or more and other spots were not
contradictory. Bottles that did not generate a MALDI ID on the first
attempt were automatically repeated; other bottles, later found to
have inconsistent results compared to VITEK® 2 results were repeated
as well. If a bottle had inconsistent results but could not be repeated it
was eliminated from consideration. If no MALDI ID was generated on
initial testing, or if there was a discrepancy compared to the reference
ID, a bottle was only processed two times.
Both bottle types were processed identically. Samples and reagents
were brought to room temperature before use.
SAMPLE PREPARATION
A 2.0 ml sample of positive blood culture broth was added to 1.0 ml
of lysis buffer (0.6% Brij-97 in 0.4M CAPS, 0.2μ filtered, pH 11.7),
vortexed for 5 seconds, and allowed to incubate for 2 minutes at room
temperature. The resulting lysate was passed in a constant stream
through a 25mm 0.45μm filter (Millipore Express PLUS®, shiny side
down) for 40 seconds. If the liquid backed up, the sample addition was
slowed in order to keep the sample application area to roughly 1 cm2.
The microbial cells remaining on the filter membrane after 40 seconds
were washed 3 times with wash buffer (20 mM Na phosphate, 0.05%
Brij-97, 0.45% NaCl, 0.2μ filtered, pH 7.2) and then three times with
deionized water. For each wash, enough buffer/water was added to
the membrane to completely cover the membrane without flowing
over. All liquid was allowed to pass through the membrane before
subsequent washes.
Once the microorganisms had been washed, they were removed by
firmly scraping the membrane with a polyester fabric-tipped microswab (Texwipe CleanTips® Swabs, cat. No. TX754B). Organisms were
then directly applied to disposable MALDI target plates (Shimadzu
Biotech, cat. No. 220-99999-FM1) and immediately covered with
22
Figure 2: Lysate Application, Completed Spot for Analysis, and manifold and reservoir
RESULTS
A total of 259 bottles were included in the study, comprising 225
monomicrobic, 28 polymicrobic, and 6 that were negative on
subculture. With all bottles included, the MALDI-TOF MS was able to
identify 72.6% of positive-flagged cultures to the species level, 19.7%
gave no ID, while 3.1% were incorrect (Table 2).
There were 225 confirmed-positive bottles containing a single
organism, and MALDI was able to identify 77.8% to the species level
and 3 organisms (1.3%) were only identified to the genus level.
17.8% of bottles processed with a single organism present did not
generate a MALDI ID, and 3.1% were incorrect (Table 1). Thus, for
monomicrobic cultures, if MALDI was able to generate an ID (185) it
was correct to the species level 94.6% of the time.
Among monomicrobic cultures, 86.4% of gram negative bacteria,
75.8% of gram positive bacteria, and 88.2% of yeasts were correctly
identified by MALDI to at least the family level (Table 3).
Twenty-eight bottles had multiple organisms; one organism of the
mixture in each of 13 bottles was identified to the species level
(46.4%), and 3 were identified only to genus level (10.7%). Eleven
bottles (39.3%) gave no MALDI ID, and 1 bottle had an incorrect
result (3.6%). No more than one organism was identified from each
bottle.
Including both monomicrobic and polymicrobic cultures, when
MALDI was able to generate an ID (202) it was correct to the species
level 93.1% of the time, and 4.0% of IDs were incorrect.
Rapid Identification of Bacteria and Yeats from Positive Blood Culture Bottles by Using a
Lysis-Filtration Method and MALDI-TOF Mass Spectrum Analysis with SARAMIS Database.
Table 1: Results of All Single-Organism Identifications
Table 2: Results from all Positive Blood Culture Bottles Processed
#
Analyzed
# Species ID
consistent
with
referenceΔ
# Genus ID
consistent
with
reference
# No
MALDI
ID
#
Incorrectv
Staphylococcus (CNS)†
64
49Ғ
0
14
1
# Correct only to Genus/Family (%)
Staphylococcus aureus (MSSA)
23
22
0
0
1
# No MALDI ID, subculture positive (%)
Staphylococcus aureus (MRSA)
22
21
0
1
0
# No Growth and No MALDI ID (%)
6 (2.3)
Escherichia coli
16
14Ғ
1
1
0
# Incorrect ID (%)
8 (3.1)
Klebsiella pneumoniae
16
16
0
0
0
Streptococcus pneumoniae
11
2
0
9
0
Organism
All Blood Culture
Bottles
# Correct Species (%)
Acinetobacter baumannii
7
3
0
3
1
Candida parapsilosis
7
7
0
0
0
Candida albicans
6
5*
0
0
1
Streptococcus agalactiae
6
5
0
1
0
Corynebacterium
5
0
0
4
1
Enterococcus faecium
5
4Ғ
1
0
0
Total # Samples
188v (72.6)
6 (2.3)
51 (19.7)
259
Thirteen of these organisms were identified from bottles with multiple organisms to the
species level. Two organisms were identified to the species level with “low discrimination”,
(the MALDI gave multiple species results, one of which was correct). Fifty-three of these
organisms were determined to have “essential agreement” to the species level. Organisms
were considered to have “essential agreement” if the MALDI ID and conventional method
were in agreement at the family and genus levels, but only the MALDI provided a species
level identification
v
Table 3: Characteristics of Monomicrobic Positive Blood Culture Bottles Processed
Candida glabrata
3
2
0
1
0
Enterobacter aerogenes
3
3
0
0
0
# Gram Negative bacteria
51/59 (86.4%)
Enterobacter cloacae
3
2Ғ
1
0
0
# Gram Positive bacteria
113/149 (75.8%)
Enterococcus faecalis
3
3
0
0
0
# Yeast
Total # Samples
Streptococcus, Viridans Group
3
2*
0
1
0
Haemophilus influenzae
2
1
0
1
0
Proteus mirabilis
2
2
0
0
0
Propionibacterium acnes
2
2Ғ
0
0
0
Salmonella sp
2
Ғ
2
0
0
0
Actinomyces meyer
1
0
0
1
0
Bacillus sp
1
0
0
1
0
0
Bacteroides fragilis
1
1
0
0
Candida sp
1
0
0
0
1
Candida tropicalis
1
1
0
0
0
Fusobacterium sp
1
1Ғ
0
0
0
Lactobacillus sp
1
0
0
1
0
Morganella morganii
1
0
0
0
1
Micrococcus sp
1
1Ғ
0
0
0
Psuedomonas sp
1
0
0
1
0
0
Psuedomonas aeruginosa
1
1
0
0
Serratia marcescens
1
1
0
0
0
Streptococcus, Group B
1
1Ғ
0
0
0
Streptococcus, Group G
1
1Ғ
0
0
0
3 (1.3)
40
(17.8)
7 (3.1)
Total (%)
225
175 (77.8)
Correct/Total (%)
15/17 (88.2%)
225
*organisms were counted as correct if they matched at family and genus levels. Organisms
that had low discrimination or essential agreement were also included in the total correct.
CONCLUSION
This study demonstrates the effectiveness of this new Lysis- Filtration
method for isolating and identifying microorganisms from positive BacT/
ALERT® blood culture bottles in a clinical setting. Approximately 80%
of monomicrobic cultures were correctly identified. The differing levels
of biomass within some positive blood cultures, as well as autolysis of
some species, may contribute to the number of bottles that were unable
to generate a MALDI result. The lysis buffer used eliminates blood
cells while leaving microorganisms intact to undergo rapid analysis by
MALDI-TOF MS.
This method is advantageous as it does not require centrifugation
and produces a clean, concentrated sample of microorganism in
less than 15 minutes.
Δ Reference ID consisted of routine laboratory workup, which was not to species level where indicated (see footnote Ғ)
v Organism (MALDI-TOF ID): CNS (Micrococcus luteus), Staphylococcus aureus (Staphylococcus epidermidis),
Acinetobacter baumannii (Klebsiella pneumoniae), Candida albicans (Moraxella catarrhalis), Corynebacterium
(Propionibacerium acnes), Candida sp. (Pichia anomala), and Morganella morganii (Proteus mirabilis)
† Two organisms identified as Staphylococcus hominis by conventional methods also included here
* C. albicans includes1 correct to species with low discrimination. Viridans group Streptococci includes 2 correct with low discrimination
Ғ
48 CNS, 2 Salmonella, and one each of all other noted organisms are in “essential agreement”. Organisms were considered to have
“essential agreement” if the MALDI ID and conventional method were in agreement at the family and genus levels, but only the MALDI
provided a species level identification
4 “PROCESSING OF POSITIVE BLOOD CULTURES WITH LYSIS-FILTRATION FOR DIRECT IDENTIFICATION BY MALDITOF MS”,
bioMérieux research use only (RUO) protocol, February 13, 2012
23
➔ ECCMID / April, 2012
London (UK) VITEK® MS IVD V1
Optimized integration of new technologies (PREVI® Isola
and VITEK® MS ) in a microbiology laboratory using the
Lean 6 Sigma methodology
J. Collard1, G. Habib1, J. Djapo Tiani1, L. Van Helleputte2, H. Palumbo2.
Laboratoire Dr. Collard, Liège, Belgium; 2bioMérieux Marcy-l’Etoile, France
1
INTRODUCTION
Three steps were distinguished for this assessment:
We are an important polyvalent private laboratory in Liege (Belgium)
where the bacteriology department represents 25% of our sample
volume (350 bacteriological samples/day).
1) Data collection:
Standard Operating Procedures (SOP) were used in the lab, activity,
type of sample sharing, positive ratio, daily arrival frequencies, TAT,
staffing.
Ten Full Time Equivalent (FTE) technicians (equivalent to 70 working
hours per day) are in charge of this department, from the sorting task
to the final validation.
The laboratory is already automatized with two UF 500i + Aution
Max for the urinary cytology and chemical tests, VITEK® 2 XL for
identifications and susceptibility tests. We decided to bring to our
lab the new available technologies to win even in efficiency and
traceability: PREVI® Isola for automatic streaking and VITEK® MS for
rapid Identification by mass spectrometry method.
2) Analyze the current situation with:
Snapshot, photo, time to process, workload, bottlenecks, waiting time,
layout with spaghetti diagram, productivity.
3) This analysis has led to a number of recommendations:
• R eorganization of the workflow in functional working cells for day 0,
day 1.
It becomes more and more important to integrate them in the most
efficient way.
OBJECTIVES
Our laboratory has decided to use the Lean 6 Sigma process prior to
the implementation of new technologies.
This new procedure is to analyze and propose an action plan and
road map for:
• A reduction of the non-added-value tasks in order to increase
productivity,
• Integrating the PREVI® Isola and the VITEK® MS in a pull and
smoothed workflow,
• Standardizing processes and methods for a streamlined workflow
and a lead time reducing with 5S and kaizen events,
• A new organization of the flow and processes,
• Improvement of Turn-Around Time (TAT) using mass spectrometry
for rapid identification,
• A possible increase in activity with the same resources (space, FTE,
instruments).
Example: a dedicated trolley for PREVI® Isola
METHODS, ANALYSIS AND RECOMMENDATIONS
• Using First In First Out (FIFO) management, single piece flow,
A performance assessment was performed during 5 days in February
2011 by a team specialized in Lean 6 Sigma methods: 1 Black belt
external consultant plus 2 Green belt bioMérieux consultants.
24
• Adapt the good balance workload / workforce,
•O
rganize new events to put in place the new organization
➔ Change management (Lab performance improvement with
KAIZEN and 5S events).
Optimized integration of new technologies (PREVI® Isola and VITEK® MS ) in a
microbiology laboratory using the Lean 6 Sigma methodology
RESULTS
CONCLUSION
1) Short term result obtained:
6 months after the laboratory workflow modification and installation
of the new automated systems, the first conclusions show:
• A reduction of TAT for the more complex samples,
• A reduction of the workload and stress for laboratory technicians,
• The ability to integrate 10% additional samples in the current
organization,
• The reallocation of a 1.5 FTE on dispatching tasks and on quality
assurance,
• A new lab layout Lean orientated.
The integration of new technologies (mass spectrometry for rapid
identification and automated plate streaking) utilizing the Lean 6 Sigma
process enabled us to optimize the whole microbiology workflow.
Day Zero Other
This enabled us to also decrease the workload, the TAT,
facilitate introduction of the accreditation process and handle
an increased volume of samples. We were able to allocate
staff in line with workload variations and build flexibility to
manage an expected demands.
The next step will be to put in place all the recommendations, the KPI
(Key Performance Indicators) for the performance measurement, the
training, and perform improvement events to achieve the middle and
long term targets.
New lab layout Lean oriented
Day Zero Urine
Day Zero Stool
Incubator
2) Middle and Long Term Targets:
The implementation of recommendations in terms of organization,
automation and training, will allow the laboratory:
• T o improve the productivity from 4.89 to 8.97 samples /working hour
(83 % productivity savings),
• T o reallocate 4 FTE to contain lab expenditure, to more added value
tasks (Accreditation), or to improve profitability by an increased
activity (50 %) with the same resources
KPI productivity TARGET 8.97 samples/working hour
KPI productivity CURRENT 4.89 samples/working hour
• To reduce Turn-Around Time (TAT)
25
Rapid and accurate identification of Campylobacter jejuni
and Campylobacter coli isolates using the VITEK® MS
MALDI-TOF mass spectrometry system
Department of Microbiology, Sandwell & West Birmingham Hospitals NHS Trust, Birmingham, United Kingdom
OBJECTIVES
RESULTS
Within the genus Campylobacter, C. jejuni and C. coli are the main
causes of human Campylobacter enteritis.
Table of Results.
In routine diagnostic laboratories, the differentiation of Campylobacter
spp. can be a challenge due to their poor biochemical activity, resulting
in many laboratories only reporting isolates to genus level.
The objective of this study was to show the easy, rapid and accurate
identification of enteritis causing Campylobacter species isolated from
clinical samples using the recently launched VITEK® MS (bioMérieux,
France) Matrix Assisted Laser Desorption/Ionisation Time of Flight
(MALDI-TOF) mass spectrometry system.
METHOD
98 previously identified strains of Campylobacter spp (49
Campylobacter coli and 49 Campylobacter jejuni), collected in 2010,
were subcultured onto Charcoal Cefoperazone Deoxycholate Agar
(C.C.D.A.) (bioMérieux, France). Strains were grown in micro-aerophilic conditions using the GENbox system (bioMérieux, France).
4 reference control strains were included, NCTC 11322, ATCC 33560
(C. jejuni) and NCTC 11366, ATCC 33559 (C. coli).
small amount of a single bacterial colony to provide a thin layer of
organism using a disposable plastic loop1.
This was then overlaid with 1μL of C.H.C.A. matrix solution (bioMérieux,
France) and allowed to air dry1. The resulting slides were then analysed in
the VITEK® MS (bioMérieux, France) instrument, using the automatic
database analysis of the obtained mass spectra within MYLA®
(bioMérieux, France) software to provide isolate identification1.
A second target spot was analysed if no spectra or no identification
was obtained.
1. Pick colony
26
3. Add Matrix
5. Review results
Total Strains
Species
ID correct
Campylobacter jejuni
49
49
Campylobacter coli
49
49
Totals
98
98
The results showed that the MALDI -TOF MS was correct in 100%
of the identifications for all 98 strains, including all reference strains.
The complete identification of all 98 strains was available in under
90 minutes.
CONCLUSIONS
The VITEK® MS MALDI-TOF system provides an easy, accurate and
importantly rapid identification of the two common enteritis causing
strains of Campylobacter species.
MALDI-TOF mass spectrometry provides an accurate alternative
to traditional identification methods.
REFERENCES:
Evaluation of the VITEK® MS MALDI-TOF mass spectrometry
system in a routine clinical laboratory.
N. Reading, A.Dadrah, A.Symonds, H.M. Kilgariff, N Ratnaraja
Department of Microbiology, Sandwell & West Birmingham Hospitals NHS Trust, Birmingham, United Kingdom
OBJECTIVES
RESULTS
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI-TOF MS) is a rapid method for identification of
microorganisms.
The VITEK® MS (bioMérieux, France) is a recently launched MALDI-TOF
MS system for rapid identification of bacterial and yeast isolates.
Our objective was to evaluate the performance of the VITEK® MS in a
routine clinical microbiology laboratory across a wide range of bacteria
and yeasts.
METHODS
A total of 617 previously isolated, well characterised strains were
tested on the VITEK® MS system.
This comprised a total of 40 different genera and 111 individual
species. 55 were reference strains obtained from ATCC or NCTC
collections. Strains were cultured onto non-selective media considered
suitable for normal growth.
small amount of test isolate to provide a thin layer of organism1. Yeast
strains were overlaid with 0.5μL of Formic Acid (bioMérieux, France),
air dried and further overlaid with 1μL of C.H.C.A. matrix solution
(bioMérieux, France) and air dried1.
Bacterial isolates were overlaid with 1μL of C.H.C.A. matrix solution
and allowed to air dry1. The resulting slides were then processed in the
VITEK® MS instrument with automatic database analysis of the obtained
mass spectra within MYLA® software2 (bioMérieux, France). A second
target spot was analysed if no spectra or identification was obtained.
Discordant isolates were subsequently identified on a VITEK® 2
(bioMérieux, France) system or by using extended phenotypic
methods and were considered to be the reference identification.
Table of results
Total Total Species ID Family/ Genus ID Mis-
No
Strains
Species correctGroupcorrectIdentified
identification
ID Correct
Enterobacteriaceae121 19
103
121
121
Non Fermenting
Gram Negative 101
16
96
96
100
1
Bacilli
Staphylococci 11210 112 112 112
Streptococci 80
18767878 2
Enterococci 607 60 60 60
HACEK Group
29
4
29
29
29
Neisseria
135 13 13 13
Moraxella
173 17 17 17
Anaerobes 24
101919221 1
Yeasts
30
9292929 1
Miscellaneous30
10252727 3
TOTALS
617
111579601608 1
8
579 out of 617 (93.8%) isolates gave a good identification to species
level. 601 out of 617 (97.4%) isolates gave an identification to group
level. Finally, 608/617 (98.5%) isolates gave a correct genus level
identification. The remaining 9 isolates failed to identify or gave an
incorrect identification profile even after multiple testing and subculture
onto alternative media.
CONCLUSIONS
The VITEK® MS MALDI TOF mass spectrometry system is a fast,
reliable method to identify clinically relevant bacterial and yeast
isolates.
Identification was available very rapidly, saving 24 hours in many
cases when compared to traditional phenotypic or biochemical identification methods.
Although the system failed to differentiate some organisms within the
same group e.g. S.mitis and S.oralis, the given group identification or
slash-line identification proved to be correct and alternative molecular
methods often struggle to delineate these strains.3
1. Pick colony
4. Add Matrix
3. Add Formic Acid
(Yeasts)
6. Review results
Mass spectrometry is a reliable method to replace traditional
bacterial identification methods in routine clinical laboratories.
REFERENCES:
2. MYLA® User Manual, bioMérieux, France 2011
3. Richard Facklam, 2002, Whatever Happened to the Streptococci: Overview of
Taxonomic and Nomenclature Changes.Clin. Microbiol. Rev. 2002, 15(4):613.
27
London (UK) Additional Research Capabilities on the VITEK® MS RUO
Clinical Testing of Bacteria and Yeast from Pediatric
Patients by Using MALDI-TOF VITEK® MS System
V. Kasinathan1, X. Zheng2, A. Fothergill1, D. Carter2, Y.F. Wang1
1
Emory University & Grady Memorial Hospital, Atlanta, GA, USA; 2 Northwestern University & Children’s Memorial Hospital, Chicago, IL, USA
ABSTRACT
Clinical isolates including yeasts and bacteria from two children’s
hospitals were used for testing by using the VITEK® MS System.
The results generated from MALDI-TOF MS that gave the definitive
identification to genus level were used for comparison with results
from conventional culture methods and additional 16S rDNA
sequencing methods for challenging organisms. Non-duplicated
clinical isolates including isolates from two children’s hospitals,
were collected from blood, spinal fluid, respiratory, wound, stool,
and urine cultures, and were used for MALDI-TOF MS testing.
With the exception of Shigella isolates, the clinical testing data
demonstrate the capability of MALDI-TOF VITEK® MS method in
correct and rapid identification of pathogenic bacteria and yeasts in
pediatric patient populations.
Table 1
Conventional
Culture Media
MALDI-TOF MS
genus
No.
species
MRSA
Staphylococcus
aureus
7
S. aureus
Staphylococcus
aureus
7
S. agalactiae
Streptococcus
agalactiae
7
E. faecalis
Enterococcus
faecalis
5
E. coli
Escherichia
coli
4
P. aeruginosa
Pseudomonas
aeruginosa
3
S. pyogenes
Streptococcus
pyogenes
3
E. faecium, VRE
Enterococcus
faecium
2
H. influenzae
Haemophilus
influenzae
2
BACKGROUND
Salmonella sp
Salmonella
sp.
2
Matrix-assisted laser desorption/ionization time-off-light mass
spectrometry (MALDI-TOF MS) can be used to detect microorganisms
rapidly from culture isolates.
S. marcescens
Serratia
marcescens
2
S. pneumoniae
Streptococcus
mitis/oralis/
pneumoniae
2
VITEK® MS System with SARAMIS database by bioMerieux is a
commercially available MALDI-TOF MS system for rapid detection of
bacterial and yeast isolates.
A. baumannii
Acinetobacter
baumannii
1
C. parasilopsis
Candida
parapsilosis
1
E. cloacae
Enterobacter
cloacae
1
E. avium
Enterococcus
avium
1
This study is designed to use the VITEK® MS to detect clinical isolates
from pediatric patients seen in one children’s hospital in the Southeast
and another in Midwest of United States.
KP ESBL
Klebsiella
pneumoniae
1
METHODS
P. mirabilis
Proteus
mirabilis
1
Clinical isolates including yeasts and bacteria from two children’s
hospitals were used for testing by using the VITEK® MS System.
The results generated from MALDI-TOF MS that gave the definitive
identification to genus level were used for comparison with results
from conventional culture methods and additional 16S rDNA sequencing
methods for challenging organisms.
P. fluorescens/putida
group
Pseudomonas
putida
1
S. pyogenes
Streptococcus
mitis/oralis/
pseudopneumoniae
1
Salmonella species
Salmonella
enterica
subsp. enterica
8
RESULTS
S. sonnei
Escherichia
coli
11
Total of 137 non-duplicated clinical isolates including 73 isolated from
one hospital (Table 1) and 64 isolated from another one (Table 2) were
collected from blood, spinal fluid, respiratory, wound, stool, and urine
cultures, and were used for MALDI-TOF MS testing.
Total
Of 64 isolates including 9 challenging organisms, only 2 organisms
could not be further identified at species level.
Among 73 isolates tested, 62 were routine isolates and 11 were Shigella
isolates. Of 62 isolates, only 1 Streptococcus pyogenes from throat
culture was identified as S. mitis, though similar isolates from other
patients were identified correctly.
Among 11 Shigella isolates tested, all were misidentified as E. coli, which
was consistent with 12 isolates from adult patients.
28
73
Clinical Testing of Bacteria and Yeast from Pediatric Patients by Using MALDI-TOF
VITEK® MS System
Table 2
Conventional
Culture Media
MALDI-TOF MS
genus
No.
species
E. coli
Escherichia
coli
13
S. aureus
Staphylococcus
aureus
13
P. aeruginosa
Pseudomonas
aeruginosa
7
E. cloacae
Enterobacter
cloacae
4
K. pneumoniae
Klebsiella
pneumoniae
4
P. mirabilis
Proteus
mirabilis
3
S. epidermidis
Staphylococcus
epidermidis
3
S. marscescens
Serratia
marcescens
3
A. xylosoxidans
Achromobacter
alcaligenaceae
2
A. xylosoxidans
Achromobacter
sp.
1
B. bronchiseptica
Bordetella
bronchiseptica/
parapertussis/pertussis
1
Diphtheroides
Corynebacterium
amycolatum/striatum
1
E. cloacae
Enterobacter
sp.
1
E. faecalis
Enterococcus
faecalis
1
K. oxytoca
Klebsiella
oxytoca
1
Micrococcus sp
Micrococcus
luteus
1
MRSA
Staphylococcus
aureus
1
S. saprophyticus
Staphylococcus
saprophyticus
1
S. maltophilia
Stenotrophomonas
maltophilia
1
S. mitis / oralis
Streptococcus
mitis/oralis/
pseudopneumoniae
1
S. anginosus
Group
Streptococcus
sanguinis
1
Total
64
CONCLUSION
With the exception of Shigella isolates, the clinical testing data
demonstrate the capability of MALDI-TOF VITEK® MS method
in correct and rapid identification of pathogenic bacteria and
yeasts in pediatric patient populations.
29
04-13 / 9305156 010/GB/A / This document is not legally binding. bioMérieux reserves the right to modify specifications without notice / BIOMERIEUX, the blue logo, API, BacT/ALERT, Myla, PREVI, SARAMIS and VITEK are used, pending and/or registered trademarks
belonging to bioMérieux or one of its subsidiaries or one of its companies / Any other name or trademark is the property of its respective owner / Photos: M. Welker / bioMérieux S.A. RCS Lyon 673 620 399 / Printed in France / THERA Conseil / RCS Lyon B 398 160 242
bioMérieux S.A.
69280 Marcy l’Etoile
France
Tel.: 33 (0)4 78 87 20 00
Fax: 33 (0)4 78 87 20 90
www.biomerieux.com
www.biomerieux.com/vitek-ms

EVOLUTION OF THE VITEK MS - bioMérieux Clinical Diagnostics

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