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Journal of Clinical Microbiology, December 2001, p. 4433-4439, Vol. 39, No. 12
Departments of
Pathology1 and
Medicine,2 National Cheng Kung
University Medical Center, and Department of Medical
Technology,3 National Cheng Kung University
Medical College, Tainan, Taiwan
Received 5 July 2001/Returned for modification 14 September
2001/Accepted 27 September 2001
Klebsiella pneumoniae strains with the transferable
carbapenem-hydrolyzing metallo- The emergence of acquired
metallo- Both IMP- and VIM-type MBLs have been detected in Taiwan (27,
28). Both IMP-1 and VIM-2 were found in Pseudomonas
putida and Pseudomonas stutzeri (27), and
a variant of the VIM-2 enzyme, VIM-3, was found in P. aeruginosa (27). A variant of the IMP-2 enzyme,
IMP-8, was identified from a clinical isolate of Klebsiella pneumoniae, which produced the SHV-12-type extended-spectrum
Bacterial strains, clinical isolates and patients.
A total
of 3,458 clinical isolates of K. pneumoniae were
consecutively collected at the National Cheng Kung University Medical Center, a 900-bed university hospital in southern Taiwan, from January
1999 to December 2000. Of these isolates, 1,622 and 1,836 isolates were
collected in 1999 and 2000, respectively. All these isolates were
identified by using the conventional techniques (5) and/or
the API 20E system (bioMérieux, Marcy l'Etoile, France). MBLs
have been shown to confer resistance to ceftazidime and cephamycins;
however, susceptibilities to carbapenems for MBL producers
covered a wide range (4, 7, 8, 10, 12, 13, 17, 19, 21-23, 27,
28, 30). Thus, only the isolates that met the following criteria
on the basis of the results of the disk diffusion method were selected
for further experiments: reduced susceptibilities to imipenem
(inhibition zone diameter, <16 mm) or meropenem (inhibition zone
diameter, <16 mm), or resistance to both ceftazidime (inhibition zone
diameter, Colony blot hybridization.
Colony blot hybridization was
performed as described elsewhere (6, 27). The DNA probes
generated by PCR amplification of the entire
blaVIM-1,
blaVIM-2,
blaIMP-1, and
blaIMP-2 were prepared as described
previously (27) and were labeled with [ PCR amplification and DNA sequencing.
Plasmids from clinical
isolates were prepared by a rapid alkaline lysis procedure
(24). PCR assays were performed to amplify the entire
sequences of the blaIMP-1-,
blaIMP-2-,
blaVIM-1-,
blaVIM-2-, blaSHV-, and
blaTEM-related genes as described
previously (27, 28). The amplicons were purified
with PCR cleanup kits (Roche Molecular Biochemicals, Mannheim, Germany)
and were sequenced on an ABI PRISM 310 sequencer analyzer (Applied
Biosystems, Foster City, Calif.). The PCR and sequencing primers used
were described elsewhere (27, 29).
blaIMP-8 and
blaVIM-3 from the control strains were
successfully amplified with the primers for
blaIMP-2 and
blaVIM-2, respectively.
Transfer of resistance.
Conjugation experiments were
performed as described previously (20, 29) with
streptomycin- and rifampin-resistant Escherichia coli C600
as the recipient (2). Tryptic soy agar plates supplemented with 500 µg of streptomycin (Sigma Chemical Company, St. Louis, Mo.)
per ml or 64 µg of rifampin (Sigma) per ml and 10 µg of ceftazidime (Glaxo Group Research Ltd., Greenford, United Kingdom) per ml were used
to select transconjugants. Plasmids from E. coli
transconjugants were digested with EcoRI (Roche Molecular
Biochemicals). Digested DNA samples were analyzed by electrophoresis on
0.8% agarose gels. The gels were stained with ethidium bromide
(Sigma), and plasmid bands were visualized under UV light. The plasmid
sizes of transconjugants were estimated by adding up restriction fragments.
Analytical IEF.
Crude preparations of Susceptibility tests.
The MICs of five PFGE analysis.
Genomic DNAs prepared by the procedure of
Piggot et al. (18) were digested overnight with 10 U of
XbaI (New England Biolabs, Beverly, Mass.) as recommended by
Tenover et al. (25) and were subjected to pulsed-field gel
electrophoresis (PFGE) with the Pulsaphor plus system (Amersham
Pharmacia Biotech) as described previously (29). DNA
fragments were separated in 1% agarose gels in 0.5× Tris-borate-EDTA
buffer at 180 V for 30 h, with pulse times ranging from 5 to
35 s. The results were interpreted according to the criteria of
Tenover et al. (25)
Screening of isolates.
On the basis of the NCCLS criteria,
only five of the 3,458 isolates showed reduced susceptibilities to
imipenem (inhibition zone diameter, <16 mm) or meropenem
(inhibition zone diameter, <16 mm). The five isolates also
demonstrated resistance to ceftazidime (inhibition zone diameter, IMP-8 producers and clinical features.
The
blaIMP-2-specific probe yielded a
strong hybridization signal with 40 of the 140 isolates in colony
hybridization experiments. Of the 40 isolates, 36 were found to carry
blaIMP-8,
blaSHV-12, and
blaTEM-1 by PCR and nucleotide
sequencing, and four were found to harbor
blaIMP-8,
blaSHV-11, and
blaTEM-1. Nine and 31 blaIMP-8-positive isolates were
collected in 1999 and 2000, respectively. The
blaVIM and
blaIMP-1 genes were not detected in the
remaining 100 isolates in both colony hybridization experiments and PCR assays.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4433-4439.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Outbreak of Infection with Multidrug-Resistant Klebsiella
pneumoniae Carrying blaIMP-8 in a
University Medical Center in Taiwan
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases, which
include IMP- and VIM-type enzymes, remain extremely rare. To
investigate whether IMP- or VIM-producing K. pneumoniae
isolates had spread at a university medical center in Taiwan, a total
of 3,458 clinical isolates of K. pneumoniae
consecutively collected in 1999 and 2000 were tested by the agar
diffusion method, colony hybridization, PCR, and nucleotide sequencing.
A total of 40 isolates (1.2%), or 17 nonrepetitive isolates, from 16 patients were found to carry blaIMP-8, a
metallo--lactamase gene recently identified from a K.
pneumoniae strain in Taiwan. Carriage of
blaVIM or other
blaIMP genes was detected in none of the
remaining isolates. Of the 17 nonrepetitive
blaIMP-8-positive isolates, 15 isolates
(88.2%) appeared susceptible to imipenem (MICs, 
4 µg/ml)
and meropenem (MICs, 1 µg/ml), indicating the difficulty in
detecting blaIMP-8 in K.
pneumoniae by routine susceptibility tests; 14 isolates
(82.4%) produced SHV-12 as well; and 14 isolates (82.4%) were also
resistant to fluoroquinolones. The organisms caused wound infections in
eight patients and bloodstream infections in three patients. They were
not directly associated with the death of nine patients. Before the
recovery of the blaIMP-8-positive isolates,
all 16 patients had undergone various surgical procedures, and 15 patients had been admitted to the surgical intensive care unit,
suggesting a nosocomial outbreak. Two major patterns were observed by
pulsed-field gel electrophoresis for 14 of the 17 nonrepetitive
isolates, indicating that the clonal spread was mainly
responsible for the outbreak.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamases (MBLs) in gram-negative bacilli is
becoming a therapeutic challenge because the enzymes usually possess a
broad hydrolysis profile that includes carbapenems and
extended-spectrum -lactams (13). Two major groups of
MBLs have been described: IMP- and VIM-type enzymes (13).
IMP-1 was the first identified acquired MBL (17) and has
spread among Enterobacteriaceae, Pseudomonas
aeruginosa, and other nonfastidious gram-negative nonfermenters in
Japan (8, 9, 22, 23). In the past three years, a number of
acquired MBLs were identified in Europe (12, 19, 21) and
the Far East (7, 10, 27, 28, 30). IMP-2 was identified
from a clinical isolate of Acinetobacter baumannii in Italy
(21). VIM-1 was identified from a clinical isolate of
P. aeruginosa in Italy (12), and outbreaks of
the VIM-1-producing P. aeruginosa isolates have been
recognized in Greece (26) and Italy (4). VIM-2 was first identified from a clinical isolate of P. aeruginosa in France (19) and has been found in
P. aeruginosa in Japan (N. Shibata, Y. Arakawa, H. Kurokawa,
Y. Doi, and K. Shibayama, Abstr. 101st Gen. Meet. Am. Soc. Microbiol.,
abstr. C-524, p. 273, 2001) and in Pseudomonas and
Acinetobacter spp. in Korea recently (K. Lee, J. B. Lim, J. Yum, D. Yong, J. R. Choi, Y. Chong, J. M. Kim, and
D. M. Livermore, Abstr. 40th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. 2003, p. 123, 2000). All acquired MBL genes found so
far were inserted in integrons (1, 7, 10, 11, 12, 19, 21, 28,
30).
-lactamase (ESBL) and TEM-1 as well (28). Reports
of MBL-producing K. pneumoniae isolates remain rare. In
Japan, only one IMP-1-producing K. pneumoniae isolate was
detected in two surveys of gram-negative bacilli (8, 23).
Outside Japan, there was only one confirmed report of an IMP-1-producing K. pneumoniae isolate collected from
Singapore (T. H. Koh, G. S. Babini, N. Woodford, L.-H. Sng,
L. M. C. Hall, and D. M. Livermore, Letter, Lancet
353:2162, 1999). Since K. pneumoniae is notorious
as a host of resistance plasmids and is one of the major causes of
nosocomial infections (5), the present study was carried
out in order to investigate the prevalence of K. pneumoniae
producing IMP- or VIM-type MBLs in a university medical center in
Taiwan. A nosocomial outbreak of K. pneumoniae carrying
blaIMP-8 in the intensive care units
(ICUs) was recognized, and thus a retrospective analysis of the cases
from which the IMP-8-producing isolates were recovered was also conducted.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
14 mm) and cefoxitin (inhibition zone diameter, 14 mm).
The medical records of the patients with MBL-producing isolates were
reviewed. The bacterial strains used as controls for colony
hybridization and PCR included blaVIM-1-containing P. aeruginosa VR-143/97 (12),
blaVIM-2-carrying P. putida
NTU-91/99 (27),
blaVIM-3-containing P. aeruginosa NTU-26/99 (27),
blaIMP-1-carrying P. putida NTU-92/99 (27), blaIMP-2-containing A. baumannii AC-54/97 (21), and
blaIMP-8-carrying K. pneumoniae KPO787 (28).
-32P]dCTP (Amersham Pharmacia Biotech, Hong
Kong, China) by the random priming technique with a commercial kit
(Gibco BRL, Life Technologies, Gaithersburg, Md.). Since there are only
two nucleotide differences between
blaVIM-2 and
blaVIM-3 (27) and four
nucleotide differences between blaIMP-2
and blaIMP-8 (28), the
VIM-3- and IMP-8-producing control strains can also be hybridized with
the blaVIM-2- and blaIMP-2-specific probes, respectively.
-lactamases were
obtained by sonication (3) and were subjected to
analytical isoelectric focusing (IEF) as described previously
(14, 27, 29). -Lactamase activity was detected by
overlaying the gels with 0.5 mM nitrocefin (Oxoid, Basingstoke, United
Kingdom) in 50 mM HEPES (pH 7.5) supplemented with 2 mM
ZnCl2 (21, 27).
-lactam agents
were determined by the agar dilution method, and the susceptibilities
to eight non--lactam antibiotics were determined by the disk
diffusion method. Both tests were performed and interpreted according
to the National Committee for Clinical Laboratory Standards (NCCLS)
(15, 16). The antimicrobial agents used for the agar
dilution tests and their sources are as follows: aztreonam,
Bristol-Myers Squibb, New Brunswick, N.J.; cefoxitin, Sigma Chemical
Company; ceftazidime, Glaxo Group Research Ltd.; cefotaxime,
Hoechst-Roussel Pharmaceuticals, Inc., Somerville, N.J.;
imipenem, Merck Sharp & Dohme, West Point, Pa.; and meropenem,
Sumitomo Pharmaceuticals Ltd., Osaka, Japan. Antimicrobial disks were
all obtained from Becton Dickinson Microbiology Systems, Cockeysville,
Md., including amikacin, chloramphenicol, ciprofloxacin, gentamicin,
ofloxacin, pefloxacin, tobramycin, and trimethoprim-sulfamethoxazole.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
14
mm) and cefoxitin (inhibition zone diameter, 14 mm). One hundred and
thirty-five isolates exhibited resistance to both ceftazidime and
cefoxitin but were susceptible to imipenem and meropenem. These
140 isolates were selected for further experiments.
-lactams and one of
them had also received meropenem.
blaIMP-8-positive isolates were
associated with wound infections in eight patients and caused
bloodstream infections in three patients. After isolation of the IMP-8
producers from blood samples, patients 7 and 8 received
imipenem and patient 14 received meropenem. Nine patients died
during the ICU stay due to multiorgan failure not directly related to
infections with IMP-8 producers.
TABLE 1.
Origins of blaIMP-8-containing
K. pneumoniae isolates and clinical characteristics of 16 patients with these isolates
Analytical IEF.
On IEF gels, all 40 IMP-8-producing isolates
had three major bands with pIs of 5.4, 7.6, and 8.2. The pI 7.6 band
probably represented the chromosomal SHV -lactamase of K. pneumoniae, the pI 5.4 band might represent the TEM-1
-lactamase, and the pI 8.2 band might represent either IMP-8 alone
or a mixture of IMP-8 and SHV-12 (29).
PFGE.
Except for two isolates from patient 13, the repetitive
IMP-8-producing isolates recovered from the same patient had identical PFGE patterns. Thus, there were 17 nonrepetitive isolates identified in
the outbreak. The PFGE results are summarized in Table
2 and are shown in Fig.
1. Five PFGE patterns were identified. Of
the 14 isolates producing both IMP-8 and SHV-12, 11 isolates had
pattern A. Isolates 3599/00, 00d401, and 99w853 had distinct patterns B, D, and E, respectively. Although isolate 00m869 exhibited a resistance phenotype different from those of isolate 3396/00 and 00t801, all three isolates had pattern C.
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Susceptibility tests.
The susceptibilities of the
nonrepetitive blaIMP-8-positive
isolates to various -lactams are summarized in Table 2. All 40 isolates were resistant to ceftazidime, cefotaxime, and cefoxitin. Thirty-six isolates, or 14 nonrepetitive isolates, which all carried blaIMP-8,
blaSHV-12, and
blaTEM1, were also resistant to
aztreonam (MICs, 
64 µg/ml), while only four isolates, or three
nonrepetitive isolates, which harbored
blaIMP-8,
blaSHV-11, and
blaTEM-1 were susceptible to this
agent (MICs, 0.06 to 0.25 µg/ml). Only five isolates, or two
nonrepetitive isolates, exhibited resistance to imipenem
(MICs, >256 µg/ml) and reduced susceptibilities to meropenem (MICs,
8 to 16 µg/ml). All the other isolates appeared susceptible to
carbapenems. The agar diffusion tests revealed that all 40 isolates studied were also resistant to non--lactam antibiotics
(Table 2).
Conjugation experiments. The 17 nonrepetitive blaIMP-8-positive isolates were subjected to conjugation experiments. The blaIMP-8-containing plasmids were successfully transferred from 13 of 17 isolates to E. coli C600. PCR and nucleotide sequencing showed that 11 transconjugants carried blaIMP-8, blaSHV-12, and blaTEM1 and that two transconjugants carried blaIMP-8 and blaTEM-1. Expression of blaIMP-8 by the transconjugants was confirmed by IEF analysis. Resistance to chloramphenicol, trimethoprim-sulfamethoxazole and aminoglycosides was also transferred to E. coli from all K. pneumoniae isolates except isolate 00m869, in which chloramphenicol resistance was not transferable (Table 2). The sizes of the plasmids transferred to E. coli were all >100 kb.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present study indicates that MBLs in K. pneumoniae remained uncommon in the university medical center. Of the 3,458 K. pneumoniae isolates, only 40 isolates (1.2%) from 16 patients were found to carry blaIMP-8. Neither the other blaIMP genes nor the blaVIM genes were detected in the remaining isolates. Nine IMP-8-producing isolates from six patients were identified among 1,622 isolates collected in 1999 (0.6%), and 31 blaIMP-8-positive isolates from 10 patients were recognized from 1,836 isolates collected in 2000 (1.7%), suggesting an increasing prevalence rate of blaIMP-8-positive K. pneumoniae in the university hospital. The increased prevalence rate could be in part due to the nosocomial outbreak.
Correlation between carriage of blaIMP-8 and of carbapenem resistance in our isolates was imperfect (Table 2). Only a total of five blaIMP-8-containing isolates, or two nonrepetitive isolates, were resistant to carbapenems. After conjugation, all E. coli transconjugants appeared susceptible to carbapenems. Similar findings have also been described in many reports of MBLs (4, 7, 8, 10, 12, 13, 17, 19, 22-23, 27, 28, 30). In addition, it is also noted that the cloned IMP or VIM enzymes confer only low-level resistance to carbapenems in E. coli (7, 10, 12, 17, 19, 21, 28, 30). Thus, there have been two speculations about the imperfect correlation: either the MBL genes are not expressed, or substantive resistance might require reduced uptake of the carbapenem as well as the presence of MBLs (13). Koh et al. (T. H. Koh, L. H. Sng, G. S. Babini, N. Woodford, D. M. Livermore, and L. M. C. Hall, Letter, Antimicrob. Agents Chemother. 45:1939-1940, 2001) described the loss of a major 39-kDa outer membrane protein in an IMP-1-producing K. pneumoniae isolate with high-level resistance to carbapenems recently, supporting the speculation that high-level resistance to carbapenems demands impermeability and an IMP enzyme. This model might also apply to our isolates. The fact that most of our blaIMP-8-positive isolates were susceptible to carbapenems indicates the difficulty in detecting K. pneumoniae isolates with the IMP enzymes by using routine susceptibility tests. Therefore, determinations of carbapenemase activity or molecular biology techniques are needed for the purposes of epidemiology and, perhaps, patients' treatment.
Monobactams are stable to hydrolysis by MBLs (7, 10, 12, 17, 19,
21, 30); however, 36 of the total of 40 blaIMP-8-containing isolates
(90.0%), or 14 of the 17 nonrepetitive isolates (82.4%), were
resistant to aztreonam. Production of the SHV-12 ESBL in addition to
IMP-8 by these isolates should be responsible for their resistance to
aztreonam. Of greater concern is that all the IMP-8-producing isolates
were also resistant to most non--lactam agents (Table 2). All of
them were resistant to at least one kind of aminoglycosides, and 37 of
the total of 40 isolates (92.5%), or 14 of the 17 nonrepetitive
isolates (82.4%), were resistant to fluoroquinolones (Table 2).
Therefore, strict infection control measures should be implemented with
the appearance of such multidrug-resistant isolates to prevent their spread.
Five patterns were obtained by PFGE. Eleven and 3 of the 17 nonrepetitive isolates exhibited PFGE patterns A and C, respectively, indicating that the nosocomial outbreak of blaIMP-8-containing K. pneumoniae was mainly due to clonal spread. The isolates from patient 14 exhibited the same PFGE and plasmid profiles as those from the surgical ICU, suggesting that the blaIMP-8-positive strain in the medical ICU might have been spread from the surgical ICU. Isolates 00d401 and 00f195, both of which were recovered from patient 13, gave different PFGE patterns, suggesting horizontal transfer of the resistance plasmid between these two isolates. Isolates 00m869, 3396/00, and 00t801 all had PFGE pattern C (Table 2). Isolates 99e843, 00f195, and 00m869 had resistance phenotypes different from those of isolates with the same PFGE patterns. The discrepancy could be due to loss or acquisition of resistance genes in the epidemiologically related isolates under various selective pressures.
In an outbreak caused by IMP-1-producing gram-negative rods at a
hospital in Japan (8), 53.8% of the IMP-1-producing
P. aeruginosa isolates were recovered from patients with
malignant diseases, suggesting that malignancy is a risk factor for the acquisition of IMP-1-producing isolates. The present study suggests that surgery is another important risk factor for the acquisition of
MBL producers. Each one of our patients had received some kind of
surgery, and eight patients (50%) had wound infections associated with
the isolation of IMP-8 producers. In contrast, only 3 of the 16 patients (18.8%) had malignant diseases. It is noteworthy that many of
our patients with wound infections stayed at the surgical ICU during
the same period (Table 2), implying that the
blaIMP-8-positive strains could have
been spread by the health care staff. Only 1 of the 16 patients (6.3%)
had received carbapenems, while 5 patients (31.3%) had
been administered extended-spectrum -lactams prior to the isolation
of the blaIMP-8-positive isolates. The
data were consistent with those obtained by Hirakata et al. (8) and with the suggestion that the selective pressure
from carbapenems was not required for the acquisition of
MBL producers. Nine of the 16 patients finally died, but none of them
died of the infections caused by
blaIMP-8-positive isolates. Serial
wound cultures indicated that
blaIMP-8-positive isolates could
colonize in the wounds for up to approximately 2 months (Table
1). These findings indicate that even though the
blaIMP-8-containing isolates were
not necessarily responsible for the high mortality of our patients, the
morbidity they caused is still a big problem. Three patients with
bloodstream infections were administered carbapenems after
the recovery of blaIMP-8-positive
isolates on the basis of the in vitro susceptibility tests. Two of them
eventually died, although they had all survived the infections. Because
of the limited numbers of patients, it is not clear whether the
favorable response was due to the treatment regimens that included
carbapenems. Despite susceptibilities of the
blaIMP-8-positive strains to
carbapenems, the use of carbapenems for the
treatment of infections with MBL-producing strains should be restricted
to prevent the possibility of emergence of more potent IMP enzymes
under the selective pressure from carbapenems.
In conclusion, the present study indicates the emergence of infections caused by blaIMP-8-positive K. pneumoniae in Taiwan. These blaIMP-8-positive isolates were all multidrug resistant and usually produced an ESBL as well. The nosocomial outbreak identified in a university hospital was largely caused by genetically related strains. Although the blaIMP-8-positive isolates were still confined to the ICUs and spread at a low prevalence rate in the university hospital, strict infection control measures against such isolates should be implemented in order to prevent their further dissemination.
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ACKNOWLEDGMENTS |
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We kindly thank G. M. Rossolini, Dipartimento di Biologia Molecolare, Sezione Di Microbiologia, Università di Siena, Siena, Italy, for provision of the IMP-2-containing A. baumannii strain AC-54/97 and the VIM-1-containing P. aeruginosa strain VR-143/97.
This work was partially supported by grants NCKUH90-031 from National Cheng Kung University Hospital and NSC89-2320-B-006-149 from the National Science Council, Republic of China.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Technology, National Cheng Kung University Medical College, No. 1 University Rd., Tainan, Taiwan 70101. Phone: 886-6-2353535, ext. 5775. Fax: 886-6-2363956. E-mail: jjwu{at}mail.ncku.edu.tw.
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Discussion
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Journal of Clinical Microbiology, December 2001, p. 4433-4439, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4433-4439.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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