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Previous Article | Next Article 
Journal of Clinical Microbiology, September 2005, p. 4402-4406, Vol. 43, No. 9
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.9.4402-4406.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Performances of VITEK 2 Colorimetric Cards for Identification of Gram-Positive and Gram-Negative Bacteria
Frédéric Wallet,
Caroline Loïez,
Emilie Renaux,
Nadine Lemaitre, and
René J. Courcol*
Laboratoire de Bactériologie-Hygiène, Centre Hospitalier Universitaire, Lille, France
Received 15 April 2005/
Returned for modification 16 June 2005/
Accepted 22 June 2005
 |
ABSTRACT
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The purpose of this study was to evaluate the new VITEK 2 identification
cards that use colorimetric reading to identify gram-positive and
gram-negative bacteria (GP and GN cards, respectively) in comparison to
fluorimetric cards (ID-GPC and ID-GNB, respectively). A total of 580
clinical isolates and stock collection strains belonging to 116 taxa
were included in the study. Of the 249 gram-positive strains tested
with both the ID-GPC and GP cards, 218 (87.5%) and 235 (94.4%) strains
were correctly identified (to the genus and species level),
respectively. Of the 331 gram-negative strains tested with the ID-GNB
and GN cards, 295 (89.1%) and 321 (97%) strains were correctly
identified, respectively. Another focus of the study was to apply the
percentages of correct identifications obtained in this study to the
list of bacteria isolated in our laboratory (32,739 isolates) in the
year 2004. We obtained 97.9% correct identifications with the
colorimetric cards and 93.9% with fluorescent
cards.
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INTRODUCTION
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The correct and rapid identification of gram-negative and gram-positive
bacteria in clinical microbiology is the first step in the
interpretation of antimicrobial susceptibility tests for correct
treatment of patients (8).
Although the VITEK 2 system (bioMérieux, Marcy l'Etoile, France)
combined with the ID-GNB and ID-GPC cards allowed an identification
within 3 h using fluorescence reading, the weakness of this
system was the breadth of its identification database, especially for
nonfermenting bacilli, such as Pseudomonas spp. and
Acinetobacter, and for gram-positive cocci, such as
Streptococcaceae
(2,
5). New cards (GP and GN
cards) will soon be available that use colorimetric
reading. These cards are suited to the VITEK 2 system, improving the
identification of nonfermenting bacteria and gram-positive cocci
(3,
4). The aims of this study
were (i) to evaluate the performances of the new colorimetric cards in
comparison to fluorimetric cards (ID-GNB, ID-GPC) to identify 580
clinical isolates and stock collection strains belonging to 116 taxa
and (ii) to determine the accuracy obtained with both readings by
applying the percentages of correct identifications obtained in this
study with the colorimetric and fluorescent cards to the list of
bacteria isolated in our laboratory in the year
2004.
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MATERIALS AND METHODS
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Strains.
A total of 580 strains were tested,
consisting of 331 gram-negative bacilli and 249 gram-positive cocci
belonging to 68 and 48 taxa, respectively. Clinical isolates were
collected over 6 months from nonconsecutive patient cultures and
selected either to obtain around 20 strains of the most frequently
isolated species or to be in agreement with the distribution of
isolates annually recovered in the laboratory. In order to have an idea
of the performance of testing for the most rarely isolated species, a
panel of 181 microorganisms was selected from the laboratory stock
collection.
Identification protocol.
All isolates were
cultured onto Columbia agar with 5% horse blood (18 to 24 h
at 35°C) to ensure purity and viability. Stock strains were
subcultured twice. Microorganisms were tested separately
with two VITEK 2 instruments: the first for fluorescence reading, the
second for colorimetric reading. The new VITEK 2 cards and the upgraded
VITEK 2 were previously described
(3). Both systems were
used according to the recommendations of the manufacturer. Bacterial
suspensions were made in 0.45% sodium chloride solution and adjusted to
a McFarland standard of 0.50 to 0.63 by using a Densicheck system
(bioMérieux). Identical inocula of each strain were tested in
parallel using fluorimetric cards (ID-GNB, ID-GPC) and colorimetric
cards (GN, GP) according to the manufacturer's instructions. When the
identification results were different between the fluorimetric and the
colorimetric cards, the strain was retested with the both methods. In
case of persistent discrepancy, the strain was identified with API
strips (ID-32 Staph, Rapid ID-32 Strept, ID-32 E, ID-32 GN, API 20NE;
bioMérieux) to resolve the identity of the strain. When there was
a mismatch between identifications obtained with both the VITEK 2 cards
and the API strips, isolate identification was determined by DNA
sequencing of the 16S rRNA (Microseq 500; Applera, Foster City, Calif.)
(1,
10) and/or sodA
(9), and/or rpoB
(7)
gene.
Quality controls.
Fifteen strains were used as quality
controls every 2 months during the evaluation. For gram-positive
bacteria, the strains were Staphylococcus saprophyticus ATCC
BAA 750, S. aureus subsp. aureus ATCC 29213,
Kocuria kristinae ATCC BAA 752, Listeria
monocytogenes ATCC BAA 751, Streptococcus thermophilus
ATCC 19258 T, S. sciuri ATCC 29061, Enterococcus
casseliflavus ATCC 700327, and S. equi subsp.
zooepidemicus ATCC 43079. For gram-negative quality control,
the strains were Klebsiella oxytoca ATCC 700324,
Acinetobacter baumannii BAA 747, Enterobacter cloacae
ATCC 700323, Ochrobactrum anthropi ATCC BAA 749, Proteus
vulgaris ATCC 6380, Shigella sonnei ATCC 25931, and
Stenotrophomonas maltophilia ATCC 17666. Each quality control
strain was tested with both the VITEK 2 fluorimetric system and the
VITEK 2 colorimetric system.
Data analysis.
Results were
separated into four groups: first-choice identification
(i.e., the determination of the genus-and-species level was the same
with both systems and was given as excellent, very good, good, or
acceptable); low discrimination (the determination resulted in a choice
among two or four species with different values of T indexing needing a
few supplemental procedures such as oxidase, motility, indole,
pigmentation, or hemolysis testing to determine the correct
identification); misidentification (the determination resulted in
incorrect identification); and no identification (the determination
resulted in doubtful, unacceptable, or unreliable identification).
Correct identification was defined as the association of first-choice
identification and low discrimination. Results were expressed in
numbers and percentages.
Two supplemental analyses of the data
were carried out. The first analysis was performed to determine the
levels of accuracy obtained with the two systems by comparing the
bacterial species identified in the year 2004 in our laboratory and
isolated five times or more to the bacterial species tested in the
study. The second analysis was to perform the same evaluation with the
17 gram-negative rods most frequently recovered from blood cultures in
the microbiology laboratories of 33 French university hospitals
(11).
|
RESULTS AND DISCUSSION
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Of the 249 gram-positive strains tested with both
the ID-GPC and GP cards and the 331 gram-negative strains tested with
both the ID-GNB and GN cards, 218 (87.5%), 235 (94.4%), 295 (89.1%),
and 321 (97%) were correctly identified (to the genus or species
level), respectively (Tables
1 and
2). A total of 33 bacteria remained unidentified with the fluorimetric
cards, whereas the colorimetric cards did not give an identification
for five strains. Regardless of their origin (combined stock collection
and clinical isolates), fermenting gram-negative bacteria were
correctly identified with both ID-GN and GN cards (97.2% and 98.7%,
respectively). In contrast, nonfermenting gram-negative bacteria were
better identified with GN cards (92.1%) than with ID-GN cards (67%).
Gram-positive bacteria were better identified with colorimetric cards
than with fluorimetric cards. For Streptococcaceae, readings
of fluorimetric and colorimetric cards gave 87.9% and 90.9% correct
identifications, respectively. For Micrococcaceae, these
readings were 87.2% and 98.3%, respectively. Our results obtained with
gram-negative bacteria were for the most part in agreement with those
reported by Funke and Funke-Kissling
(3). Testing 511
fermenting and 144 nonfermenting gram-negative bacilli, these authors
(3) obtained slightly
better results with the GN cards than we did in the present study
(99.5% and 98.7%, respectively). Recently, in another study focusing on
gram-positive bacteria, Funke and Funke-Kissling
(4) obtained correct
identification of Streptococcaceae (217 strains) and
Micrococcaceae (147 strains) for 99.1% and 99.3% of these
bacteria, respectively, whereas the results were 90.9% and 98.3% in our
study. It should be noticed that the results reported by Funke and
Funke-Kissling (3,
4) appeared better but
that the numbers of taxa tested (13, 18, and 12 for
Micrococcaceae, Streptococcaceae, and nonfermenting
bacilli, respectively) were lower than in our study, except for
fermenting bacteria (42 taxa in both studies). In fact, as Funke and
Funke-Kissling have previously claimed to have done
(3,
4), we have selected rare
bacteria isolated in routine practice and some of them, such as S.
constellatus or S. gordonii, while infrequently isolated
in routine testing, were isolated in large numbers in our
study (for example, eight strains of S.
constellatus instead of one in Funke's study).
The GP and GN
identification cards contain new tests (16 and 21 tests for GP and GN,
respectively) allowing an improvement of the VITEK 2 databases; in
fact, 57 and 38 new species of gram-positive cocci and gram-negative
bacilli were added to the database. Of these, seven gram-positive
species (eight strains) were tested and only one strain of
Pediococcus pentosaceus was not correctly identified. Of the
17 new gram-negative species tested (36 strains), only four species
(four strains) were not correctly identified: Bordetella
bronchiseptica, Pseudomonas alcaligenes, and P.
putida were misidentified (one strain each), and one strain of
P. fluorescens was not identified. Thus, the misidentification
percentages obtained with the colorimetric cards ranged from 2.1% to
4.8%, results which were slightly better than those obtained with the
fluorescent cards (5.1% to 6.8%). These misidentified bacteria were
observed only with Streptococcus spp. such as S.
constellatus (three strains) or S. gordonii (two strains)
or nonfermenting gram-negative bacilli such as Acinetobacter
spp. belonging to genomospecies 1, 2, 3, or 13 (A. calcoaceticus/A.
baumannii complex). Compared to previous study results
(2,
5,
6), the database was
enlarged and improved, especially for nonfermenting bacilli and
Micrococcaceae. The database was also enlarged to include some
gram-positive bacilli such as Erysipelothrix rhusiopathiae and
six species of the Listeria genus.
The second aim of
this study was to evaluate the performance of the new colorimetric
cards in routine practice. Thus, we applied the percentages of correct
identifications obtained in this study with the colorimetric and
fluorescent cards to the list of bacteria isolated in our laboratory in
the year 2004. From the species included in the database of the VITEK
2, 71 species were selected, representing 32,739 bacteria isolated five
times or more in 2004. An overall correlation of 97.9% correct
identifications for gram-positive and gram-negative bacteria was
obtained, whereas it was equal to 93.9% with fluorescent cards. The
same determination was performed with a selection of 17 gram-negative
taxa isolated more frequently in 33 French university hospitals
(11). Identification with
colorimetric cards gave an overall identification to the species level
of 99.7%, whereas it was equal to 95.9% with fluorescent
cards.
The VITEK 2 system, equipped for colorimetric reading of
the new GP and GN cards, keeps the advantages of the VITEK 2
(2,
5,
6,
8), i.e., reliable
identification, fully automated incubation and interpretation, and
minimal supplemental testing required. The results provided
by the colorimetric VITEK 2 may be considered accurate due to the
improvement and the extension of its database, mainly for nonfermenting
bacteria and Streptococcaceae. In this study, the
identifications of bacteria were provided between 5.2 h
(fermenting bacteria) and 6.7 h (nonfermenting bacteria),
which was slightly greater than the time required for reading with
fluorescent cards. However, the results were always provided within a
day. In conclusion, the results obtained in this study demonstrate the
good performances of the new VITEK 2 cards, allowing their use in
routine practice with a highly acceptable level of identification
accuracy.
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ACKNOWLEDGMENTS
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We thank bioMérieux
for kindly providing the VITEK 2 system and Geneviève Bossy and
Marie-Christine Saccomani for their technical
assistance.
|
FOOTNOTES
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* Corresponding author. Mailing address: Laboratoire de Bactériologie-Hygiène, Hôpital A. Calmette, F-59037 Lille Cedex, France. Phone: 33 320 444 908. Fax: 33 320 444 895. E-mail: rcourcol{at}chru-lille.fr. 
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Journal of Clinical Microbiology, September 2005, p. 4402-4406, Vol. 43, No. 9
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.9.4402-4406.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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Abstract
Introduction
