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Journal of Clinical Microbiology, December 2000, p. 4580-4585, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Fluorescent Amplified-Fragment Length Polymorphism Genotyping
of Neisseria meningitidis Identifies Clones Associated
with Invasive Disease
Jonathan N.
Goulding,1
John V.
Hookey,1
John
Stanley,1
Will
Olver,2
Keith R.
Neal,3
Dlawer A. A.
Ala'Aldeen,2 and
Catherine
Arnold1,*
Molecular Biology Unit, SBVL, Central Public
Health Laboratory, London NW9 5HT,1 and
Meningococcal Research Group, Division of
Microbiology,2 and Department of Public
Health Medicine and Epidemiology,3 Queen's
Medical Centre, Nottingham, United Kingdom
Received 12 May 2000/Returned for modification 10 July
2000/Accepted 3 October 2000
 |
ABSTRACT |
Fluorescent amplified-fragment length polymorphism (FAFLP), a
genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from
an outbreak in a British university; 9 were from a recent outbreak in
Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal
disease; 26 were from the National Collection of Type Cultures; 58 were
carrier isolates from Ironville, Derbyshire; 1 was a disease isolate
from Ironville; and five were representatives of invasive clones of
N. meningitidis. FAFLP analysis results were compared with
previously published multilocus sequence typing (MLST) and pulsed-field
gel electrophoresis (PFGE) results. FAFLP was able to identify
hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar
but distinct by FAFLP. The results suggest that high resolution of
N. meningitidis for outbreak and other epidemiological
analyses is more cost efficient by FAFLP than by sequencing procedures.
| |
INTRODUCTION |
Neisseria meningitidis is
an obligate human pathogen of worldwide significance whose effects
range from asymptomatic carriage to lethal systemic infection. Its
ability to cause outbreaks with significant mortality is the focus of
considerable public health concern. Meningococci are carried
asymptomatically in the nasopharynxes of up to 15% of the population,
and all age groups, but especially children, may contract meningococcal
septicemia or meningitis. Fatality is high even with antibiotic and
supportive therapy.
The meningococcus is an antigenically complex bacterium with multiple
genetic mechanisms for initiating changes to its cell surface to evade
host immune defenses. Thirteen serogroups are recognized on the basis
of capsular polysaccharide antigens, five of which (A, B, C, Y, and
W-135) are commonly associated with disease. Antigenic diversity in the
PorB and PorA outer membrane proteins defines serotypes and
serosubtypes, respectively. Meningococci are transformable, and there
is frequent lateral transfer of antigen-encoding genes (capsular switching).
The population genetics of N. meningitidis have been
analyzed by multilocus enzyme electrophoresis (MLEE) (4)
and, more recently, by multilocus sequence typing (MLST)
(16). MLST-based measurement of the selectively neutral
variation that slowly accumulates in the meningococcal population shows
that the species has a very complex population structure. It is largely
panmictic, i.e., nonclonal, but it contains transient clones of
variable stability. Meningococcal strains with an increased attack rate
tend to arise by random assortment and horizontal gene transfer of the
alleles that determine disease-causing propensity (15, 22).
MLEE has identified several of these hypervirulent, hyperendemic
electropherotypes (ETs) or complexes of related N. meningitidis strains associated with disease. These include ET37,
ET5, and the A4 complex.
Sensitive and reproducible meningococcal typing methods are required
not only for population genetic and epidemiological investigations but
also for vaccine-related studies. It is necessary to identify outbreaks
associated with particular serogroups (as vaccines are serogroup
specific), to demonstrate epidemiological links between cases or
between cases and carriers in an outbreak, to monitor the changing
epidemiology of disease, and to evaluate new vaccines. Phenotypic
typing methods used to examine isolates for characteristics below the
species level, such as serogrouping, suffer from several problems,
including antigenic variability, poor expression or masking of surface
antigens, the inability to subtype all isolates, and the need to
continually enlarge the reagent panel. Molecular typing methods for
meningococci such as MLEE, MLST based solely on housekeeping genes
(seven-locus MLST), and pulsed-field gel electrophoresis (PFGE) achieve
discrimination in different ways. MLEE and seven-locus MLST are methods
based on variations that accumulate very slowly and are suitable for
long-term and global epidemiology. PFGE and other methods based on the
selection of highly variable regions of the genome which include
appropriate restriction enzymes or PCR priming sites identify the
microvariation that is required to distinguish between strains
circulating within a geographical location. For meningococci, PFGE
provides greater discriminatory power than does serology for
epidemiological investigation (2, 3). Furthermore, MLST
which includes sequences of two variable antigen genes (9-locus MLST)
is able to distinguish between strains identical by other molecular
methods such as PFGE (7).
Amplified fragment length polymorphism (AFLP) analysis is a PCR-based
genome sampling technique that reproducibly generates a specific
profile for each bacterial clone. First described by Vos et al.
(21), AFLP is emerging as a convenient tool for the study of
genetic diversity (6, 10-14). In the fluorescent AFLP (FAFLP) format for the MLEE-defined EcoR reference collection of 72 Escherichia coli strains, AFLP yields groupings nearly
identical to those of MLEE (and, by implication, those of MLST)
(1).
As FAFLP has also been used successfully for the investigation of
outbreaks of Streptococcus pyogenes and Staphylococcus
aureus (5, 9), it appears that FAFLP might be of
general use for the study of micro- and macrovariation between
bacterial strains, including effectiveness in outbreak investigations
and studies of the population genetics of N. meningitidis.
We have therefore determined the congruence of FAFLP with other
molecular typing methods, namely, MLST and PFGE, for N. meningitidis and compared them for efficiency. We have analyzed
strains from two outbreaks of meningitis previously characterized by
MLST and PFGE and 58 N. meningitidis isolates from carriers
in the village of Ironville, Derbyshire, United Kingdom. This village,
population 1,600, has recently (between August 1997 and August 1999)
experienced a protracted outbreak of invasive meningococcal disease,
with five confirmed and seven probable meningitis cases. The patients
were between 3 and 9 years old.
| |
MATERIALS AND METHODS |
Strains.
A total of 123 isolates of N. meningitidis were examined. Thirty-three were disease causing or
disease associated (from asymptomatic patient contacts). They included
nine from the 1997 Southampton University outbreak in the south of
England (S1 and S3 to S10) and nine from the 1999 Pontypridd outbreak
in Wales (P1 and P3 to P10); 15 were from epidemiologically unrelated
meningococcal disease (prefixed X) and were isolated between 1990 and
1993. Twenty-six strains, dating from 1934 to 1987, were from the
National Collection of Type Cultures (NCTC; prefixed N). Fifty-eight
carriage isolates were from throat swabs of healthy individuals from
Ironville (no prefix). Screening of the population had been undertaken
in May 1998 to ascertain the serogroup, serotype, and serosubtype of
carrier strains, and 116 people in Ironville (approximately 7.25% of
the population) were found to carry N. meningitidis
asymptomatically. Five strains of known MLEE type 37 were also
analyzed. These were one disease isolate from Ironville, isolated in
1997 (Ironville); one from the north of England, isolated in 1999 (ET37); one from Mali, isolated in 1989 (ET37a); one from Israel,
isolated in 1988 (ET37b); and one from Scotland, isolated in 1990 (ET37c). One known MLEE type five (ET5) strain, isolated in Norway in
1982, was also analyzed.
Serotyping.
Isolates were serogrouped and serotyped by the
Meningococcal Reference Unit (Manchester Public Health Laboratory)
using a standard reference collection of monoclonal antibodies
(8).
Genomic DNA extraction.
Bacteria were grown for 24 h on
chocolate agar at 37°C in a 5% CO2 atmosphere. Stock
cultures were maintained in 16% (vol/vol) glycerol broth on Preserver
Beads (Technical Services Consultants, Cambridgeshire, United Kingdom)
at 
70°C. Genomic DNA was extracted from plate cultures as follows.
One-half loopful (10 µl) of bacterial growth was removed from a
chocolate agar plate by sweeping an inoculating loop over the agar
surface. Cells were suspended in Tris-EDTA-glucose, centrifuged at
7,500 × g, resuspended in 100 µl of lysozyme, and
incubated at 37°C for 1 h. Cell wall debris, polysaccharides,
and proteins were selectively precipitated with cetyltrimethylammonium
bromide, and DNA was recovered from the supernatant by isopropanol precipitation.
FAFLP analysis.
Genomic DNAs were purified by ethanol
precipitation using standard methods, and the pellet was then
resuspended in 15.8 µl of water.
Genomic DNAs were digested in a 20-µl (total volume) mixture
containing 500 ng of DNA, 4 U of MseI (New England Biolabs
[NEB]), 2 µl of 10× MseI buffer (NEB), 0.2 µl of
bovine serum albumin (NEB), and 1 µl of RNase A (10 mg/ml) that was
incubated for 2 h at 37°C. Five units of EcoRI (Life
Technologies) was then added, along with 1.68 µl of Tris buffer (pH
7.6) and 2.1 µl of NaCl (0.5 M) to give a final volume of 25 µl and
incubated at 37°C for 2 h. Five microliters of EcoRI
adapter (2 µM), 5 µl of MseI adapter (20 µM), 5 µl
of ligase buffer (NEB), and 0.1 µl of T4 DNA ligase (40 U/µl of
NEB) were added in a final volume of 50 µl, and the mixture was
incubated at 12°C overnight. The enzyme was denatured at 65°C for
20 min, and the reaction mixture was stored at 20°C until use. The
sequence of the EcoRI adapter was: 5'-CTCGTAGACTGCGTACC CATCTGACGCATGGTTAA-5'
The sequence of the
MseI adapter
was: 5'-
TACTCAGGACTCATC GAGTCCTGAGTAGCAG-5'
PCR was performed in a final volume of 20 µl,
consisting of 2 µl of 10× PCR buffer, (Life technologies), 1.5 mM
MgCl
2, 0.5
U of
Taq polymerase, 200 µM
deoxynucleoside triphosphates, 0.25
µM Mse-O primer, 0.1 µM Eco-T
primer, and 2 µl of stored postligation
mix. The primer sequences
were as follows: Mse-0, 5'-GATGAGTCCTGAGTAA-3';
Eco-T,
5'-GACTGCGTACCAATTCT-3'. The Eco-T primer (MWG
Biotech)
was fluorescently labeled with 5-carboxyfluorescein.
Amplification
was performed on a PE9600 with predenaturation at 94°C
for 2 min,
followed by 30 cycles of denaturation at 94°C for 20 s, a 30-s
annealing step, and extension at 72°C for 2 min. The
annealing
temperature for the first cycle, 66°C, was subsequently
reduced
by 1°C for the next nine cycles, with the remaining 20 cycles
at an annealing temperature of 56°C. This was followed by a 30-min
incubation at 60°C. PCR amplicons (FAFLP profiles) were separated
on
a 377 automated sequencer (PE Biosystems) using 36-cm plates
and a 5%
SinGel (FMC) at 3 kV and 51°C for 2.5 h with an internal
molecular size marker (ROX 500; PE
Biosystems).
The output gel file was analyzed and the bands were sized using the
software package Genescan (PE Biosystems). For analysis
of fragments,
the presence or absence of which was confirmed by
eye, files were
exported to Genotyper (PE Biosystems). Eight isolates
were used as a
control for both digestion-ligation and PCR. One
hundred samples were
analyzed more than once, and the profiles
were compared to test
reproducibility. Data were translated into
binary in Excel, showing the
presence or absence of a band, and
exported into a custom-designed
software application that calculated
Dice coefficients between pairs of
isolates (
17). These data
were exported into Neighbor, a
part of the Phylip package which
uses the unweighted pair group method
with arithmetic averages
(UPGMA) to construct a tree by successive
clustering using an
average-linkage method of
clustering.
| |
RESULTS |
FAFLP reproducibility.
One hundred of the isolates were
examined twice. All duplicates gave FAFLP profiles identical to those
of the originals. Gel and amplification controls always gave identical
profiles. Figure 1 shows an example of
the reproducibility of the FAFLP analysis of meningococcus where five
FAFLP replicates of strain NCTC 8554 yield identical FAFLP profiles.
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FIG. 1.
Genotyper output of FAFLP analysis of replicates of NCTC
type strain 8554 in the range of 210- to 310-bp fragments. The five
traces represent separate FAFLP reactions on the same DNA extract, run
on different gels. The boxed numbers under the peaks of the traces are
the fragment sizes in base pairs assigned by comparison with the
standard curve generated with the internal size standard.
|
|
FAFLP fragment amplification patterns.
A total of 131 distinct
DNA fragments were amplified from the genomes of 123 meningococcal
isolates analyzed in this study. Fragments in the size range of 100 to
306 bp were used for analysis as a small number of isolates gave
suitable signal strength for analysis only within this size range. The
FAFLP profiles of individual isolates contained between 18 and 39 bands. For every isolate, three distinct DNA fragments were always
amplified; i.e., they were present in every FAFLP profile. The FAFLP
profiles yielded the tree shown in Fig. 2
and 3. Tree-building programs using
algorithms different from UPGMA, including parsimony, gave trees with
the same topology (data not shown).
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FIG. 2.
Tree representing FAFLP data for 119 N. meningitidis isolates with the serogroup, where known, indicated
for each isolate (see key). One serogroup, W-135, clustered closely by
FAFLP (circled), an exception being Ironville isolate 560.
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FIG. 3.
Tree identical to that in Fig. 2 except that the
invasive disease-causing isolates and the carriage isolates are
indicated. FAFLP separated the disease-causing strains into five
clusters (circled). The largest of these, cluster 1, consisted of the
majority of Southampton and Pontypridd, United Kingdom (UK), outbreak
strains.
|
|
FAFLP association with serogroup profiles.
Figure 2 shows the
relationship of serogroup with genotype. Only one serogroup, W-135
(circled in Fig. 2), clustered closely by FAFLP, an exception being
Ironville isolate 560, placed at the 2 o'clock position among carriage
strains of various serogroups.
Isolates of serogroups B and C were found throughout the tree. There
were certain regions where serogroup B isolates (in green)
were more
prevalent
for example, at 9 to 12 o'clock in Fig.
2.
Serogroup C
isolates (in red) were most prevalent at the 3 to
6 o'clock
position.
FAFLP of disease-causing strains.
The disease-causing isolates
of N. meningitidis formed five distinct clusters, circled in
Fig. 3. (Figures 2 and 3 are identical in form and content except for
color coding to show serogroup and epidemiological context,
respectively.)
Cluster 1 in Fig.
3 consists of the majority of the disease strains.
Five are apparently epidemiologically unrelated disease
isolates
(prefixed X). Eight are from the Southampton University
outbreak (S1
and S3 to S9, from patients and carriers. Seven are
from the Pontypridd
outbreak (P1 and P3 to P10, from patients
and carriers). One is a
disease-associated NCTC strain (12087)
deposited in 1987. Five strains
are of a known MLEE type defined
as being part of the ET37 complex of
N. meningitidis (Ironville
and ET37 to ET37c). Also found in
cluster 1 are five isolates
from the Ironville carriage study (180, 373, 383, 502, and 526).
All of these Ironville isolates belonged to
serogroup C except
502, which belonged to serogroup B. All of the
strains and isolates
in cluster 1 belong to serogroup B or C. The
remainder of the
Ironville carriage isolates are distributed throughout
the tree,
with the majority of isolates having unique
profiles.
In the Southampton University outbreak, S1, S3, and S5 had the same
FAFLP profile, as did isolates S4 and S9. Isolates S6,
S7, and S8
had unique profiles. All of these isolates were in
FAFLP cluster
1. S10, by contrast, was very dissimilar by FAFLP
analysis, being among
carriage strains positioned just to the
left of cluster 2. The nine
isolates from the Southampton University
outbreak included four
meningitis isolates (S1, S3, S4, and S5),
a contact of the patient who
was the source of S4 (S9), and four
asymptomatic-carriage isolates (S6,
S7, S8, and S10). Table
1 compares FAFLP
with other genotypic and phenotypic methods used
to analyze the
Southampton University isolates, as determined
by Feavers et al.
(
7). As noted above, FAFLP analysis generated
two profiles
among the five meningitis isolates. Isolates S1,
S3, and S5 shared a
profile, and this had a single 223-bp fragment
difference from the
profile of meningitis isolate S4 and patient
contact isolate S9. This
fragment difference corresponded to observed
differences between these
two profiles with respect to phenotype
(C:NT:P1.5 versus C:2a:NT), the
porB allele (2-37 versus 2-36),
and the
porA
allele (5a,10d versus 5,2).
Analysis of the Pontypridd outbreak (Fig.
3, cluster 1) showed that
isolates P1, P3, P6, P7, P8, P9, and P10, all causing
invasive disease,
were identical by FAFLP, although the signal
intensity of some
fragments varied between isolates. By contrast,
P4, from a patient
suspected of being infected, and P5, from a
carrier, were dissimilar
from each other and from the other Pontypridd
isolates. Table
2 compares FAFLP with other genotypic and
phenotypic
methods for the Pontypridd isolates. Six disease isolates
from
this outbreak had the same MLST profile (profile 11; Table
2),
and
all except P4 were of serotype C:2a:P1.5. Asymptomatic-carriage
isolates P3 and P8, which are included in this FAFLP cluster 1,
were
also of this serotype. Pontypridd outbreak isolate P4 was
of serotype
C:1:P1.15 and had a unique MLST profile. Carriage
isolate P5 was of
serotype C:NT:P1.14 and had a unique MLST profile.
By PFGE, all but two
(P9 and P10) of these Pontypridd isolates
had identical
macrorestriction profiles. Two of the three distinct
FAFLP profiles
found for the Pontypridd isolates corresponded
to the distinct
phenotype of P5, and the other was found for disease
isolate P4. P4 and
P5 had 10 and 13 amplified fragment differences,
respectively, from the
FAFLP profile shared by the other seven
isolates.
FAFLP cluster 2 (Fig.
3) contained five epidemiologically unrelated
meningitis isolates (prefixed X) and NCTC strains 12085
and 12086. It
also contained a representative of the hypervirulent
and hyperendemic
ET5 clone complex isolated in Norway in 1982
and initially identified
by MLEE. All but one of the isolates
in cluster 2 was known to belong
to serogroup B, except for ET5,
whose serogroup was
unknown.
FAFLP cluster 3 consisted of three isolates from the
epidemiologically unrelated disease-causing (X) collection of isolates
(X6, X15, and X17). Of these three, two were of serogroup C and
one was of serogroup B. Clusters 4 and 5 consisted entirely of
disease-causing NCTC isolates dating from 1934 to
1987.
Epidemiologically unrelated disease-causing isolates were found in
clusters 1, 2, and 3. One of these isolates, X13, had the
same FAFLP
profile as isolates S4 and S9 from the Southampton
University
outbreak.
| |
DISCUSSION |
Congruence of FAFLP with MLST.
The development of 9-locus MLST
(7), which includes sequences from two variable antigen
genes together with sequences from selectively neutral housekeeping
genes has, for the first time, enabled both the macro- and
microvariation of N. meningitidis to be analyzed using the
same method. Previously, global epidemiology (macrovariation) of
isolates has been studied using MLEE (and subsequently MLST based on
housekeeping genes) while outbreak investigation (microvariation) of
isolates has been done using a method such as PFGE. A further advantage
of MLST over other methods is its portability between laboratories
(16).
Data from this study show that FAFLP has the same high level of
resolution for the Southampton University outbreak investigation
as
9-locus MLST as determined by Feavers et al. (
7). FAFLP
also
clusters together hypervirulent lineages such as ET37 and
ET5 in the
same way as MLST. All of the Southampton University
outbreak isolates
except S10 were in a single FAFLP cluster (
1)
and were
identified as belonging to the ET37 complex by MLST (
7).
Similarly, all of the Pontypridd outbreak isolates except P4 and
P5
were in FAFLP cluster 1 and the ET37 complex by MLST (data
not shown).
Two isolates related to, but not part of, FAFLP cluster
1 (N12083 and
N8554) grouped away from cluster 1 and were not
assigned to the ET37
complex by MLST (data not
shown).
Thus, insofar as FAFLP cluster 1 corresponds to ET37, FAFLP (a
technique that samples throughout the genome, examining both
variable
and nonvariable regions, as demonstrated by predictive
modeling
experiments with a known genome sequence [
1]), has
been shown to be capable of identifying an MLST-MLEE-defined clone.
Further evidence that FAFLP can identify significant MLST-MLEE
clones
is provided by data from FAFLP cluster 2. This cluster
consisted of
five epidemiologically unrelated disease isolates,
two recent
disease-causing NCTC strains isolated in the 1980s,
and one isolate
obtained in Norway in 1982 that is representative
of ET5, another
hypervirulent lineage. These findings on meningococcal
MLEE clones
agree with our previously published FAFLP analysis
of the EcoR
reference collection, which represents the genetic
diversity of 72
E. coli strains. In that study, FAFLP was fully
congruent
with the five MLEE-defined phylogenetic groups of
Escherichia coli for that collection of strains (
1).
Olive et al. recently published costs for DNA-based typing of microbial
organisms (
18). Using their calculations, the estimated
cost
for one FAFLP reaction is $20 whereas the cost for carrying
out 9-locus
MLST on a single isolate, which in our study gave
the same amount of
information as a single FAFLP reaction, is
$360. Thus, FAFLP is much
more cost efficient than 9-locus MLST.
The setup costs for FAFLP and
MLST ($45,000 to $130,000) are the
same (
18).
Congruence of FAFLP with PFGE.
The data from both FAFLP and
PFGE for the two outbreak investigations studied here were comparable,
but PFGE was unable to discriminate between two similar but distinct
strains from the Southampton University outbreak. FAFLP had
greater resolution than PFGE for the Pontypridd outbreak. The
increased degree of resolution achieved by FAFLP can be attributed to
the double restriction enzyme digestion employed by FAFLP, as opposed
to the single enzyme digestion used in PFGE, and the consequently
higher number of DNA fragments analyzed by FAFLP (23). The
cost of a single PFGE reaction is estimated at $22, compared with $20
for FAFLP. The setup costs for PFGE are less, only $10,000 to $20,000
(18).
FAFLP association with serogroup profiles.
It is known that
ET37 complex strains are mainly of serogroup C, but serogroup B
isolates of this complex are increasingly being isolated from patients,
and recent studies have demonstrated that meningococci are capable of
capsular switching among serogroups B, C, and W-135 in vivo while
retaining hypervirulence capabilities (19, 20). Evidence for
possible capsular switching in this study is best illustrated by the
very closely related Ironville ET37 strains, of which four belong to
serogroup C and one belongs to serogroup B. As no suitable serogroup B
vaccine is available, it is important to monitor the present national
vaccination campaign in the United Kingdom for capsular switching of
hypervirulence complexes to serogroup B due to selective pressure.
Further evidence of recent capsular switching is shown by serogroup
W-135, which is fairly tightly clustered by FAFLP, apart from one
strain quite distinct from the rest.
In summary, to distinguish outbreak isolates from unrelated but
temporally and geographically proximate cases, rapid and accurate
characterization of meningococcal isolates is needed. We
believe
that this need can be met by FAFLP analysis, shown here to cost
effectively establish the identity of strains and their genetic
relationships. In the present study, FAFLP was congruent with
the
established genotypic method of MLEE or MLST and distinguished
between carriage and invasive isolates, as MLST did. The technique
has
the potential to identify hypervirulent lineages defined by
ET or MLST.
Thus, in addition to identifying microvariation that
will distinguish
strains circulating within a geographic location
in a more efficient
manner than MLST, FAFLP can simultaneously
contribute to the wider
understanding of the population genetics
and global epidemiology of the
meningococcus.
| |
ACKNOWLEDGMENT |
We thank the Meningococcal Reference Unit (Manchester Public
Health Laboratory), especially Steve Gray, for kindly supplying PFGE data.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Biology Unit, SBVL, Central Public Health Laboratory, 61 Colindale
Ave., London NW9 5HT, United Kingdom. Phone: 44 20 8200 4400. Fax: 44 20 8200 1569. E-mail: carnold{at}phls.nhs.uk.
| |
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Journal of Clinical Microbiology, December 2000, p. 4580-4585, Vol. 38, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
