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Journal of Clinical Microbiology, March 1999, p. 538-543, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparative Analysis and Serovar-Specific
Identification of Multiple-Banded Antigen Genes of
Ureaplasma urealyticum Biovar 1
Fanrong
Kong,1,2
Xuejun
Zhu,1
Weizhen
Wang,1
Xiangzhao
Zhou,1
Susanna
Gordon,2 and
Gwendolyn
L.
Gilbert2,*
Department of Dermatology, First Hospital of
Beijing Medical University, Beijing 100034, People's Republic of
China,1 and
Centre for Infectious
Diseases and Microbiology, Institute of Clinical Pathology and
Medical Research, Westmead, New South Wales,
Australia2
Received 20 July 1998/Returned for modification 20 August
1998/Accepted 2 November 1998
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ABSTRACT |
Ureaplasma urealyticum is a causative agent of
nongonococcal urethritis and is implicated in the pathogenesis of
several other diseases. The species is divided into 14 serovars and two
biovars, of which biovar 1 is most commonly isolated from clinical
specimens. Reported associations between individual serovars and
diseases have been difficult to confirm because of practical
difficulties with serotyping. The multiple-banded antigen (MBA) is the
predominant U. urealyticum antigen recognized during
infections in humans and probably has a significant role in virulence.
The 5' end of the MBA gene is relatively conserved but contains biovar,
and possibly serovar, specificity. The 5' ends of the MBA genes of standard strains of U. urealyticum biovar 1, consisting of
serovars 1, 3, 6, and 14, were amplified, cloned into pUC19, and
sequenced to identify serovar-specific differences. The 5' end of the
MBA gene sequence of serovar 3 was identical with the previously
published sequence and differed by only three bases from that of
serovar 14. Significant differences between the MBA gene sequences
allowed biovar 1 to be divided into two subgroups, containing serovars 3/14 and serovars 1 and 6, respectively, using primers UMS-125-UMA269 and UMS-125-UMA269'. Serovars 1 and 6 were distinguished by
restriction enzyme analysis of the amplicon and/or by PCR specific for
serovar 6. These methods were used to identify and type U. urealyticum in 185 (46.3%) of 400 genital specimens from women.
Biovar 1 was detected in 89.2% and biovar 2 in 18.3% of positive
specimens. Of 165 specimens containing U. urealyticum
biovar 1, 22.2% contained more than one serovar and 46.7, 46.1, and
25.5% contained serovars 1, 3/14, and 6, respectively. U. urealyticum was found in a significantly higher proportion of
pregnant women than in sex workers and other women attending a sexually
transmissible diseases clinic (P < 0.01). The methods
described are relatively rapid, practicable, and specific for
serotyping isolates and for direct detection and identification of
individual serovars in clinical specimens containing U. urealyticum biovar 1.
| |
INTRODUCTION |
Ureaplasma urealyticum is
a recognized cause of urethritis and disseminated infection in
immunocompromised patients (8, 15-17). It has also been
implicated in other genitourinary syndromes (10) and a
number of complications of pregnancy, including chorioamnionitis, preterm birth, and chronic neonatal lung disease (1-3).
However, it is found commonly among the genital tract flora of healthy women, and its pathogenic role in a small proportion of individuals is
difficult to prove (4, 23).
There are two biovars and 14 serovars of U. urealyticum. The
majority of human isolates of U. urealyticum belong to
biovar 1 (1, 6, 7, 9, 23), which includes serovars 1, 3, 6, and 14. Some serovars have been associated with disease syndromes more
often than they are found in normal flora (4, 9, 10). However, these findings are difficult to confirm because of problems with conventional serotyping methods (4, 9, 12). A rapid molecular method for identification of individual serovars would be of
great value in studies of epidemiology and pathogenesis of infections
with U. urealyticum (11, 13, 18, 23).
The MBA (multiple-banded antigen) is the predominant antigen recognized
during U. urealyticum infections and is probably an important virulence determinant. It is species specific and contains both serovar-specific and cross-reactive epitopes (19,
21-23). The 5' end of the MBA gene is relatively conserved, but
it also includes biovar- and serovar-specific regions (22).
The aim of this study was to identify sequence differences among the 5' ends of the MBA genes of U. urealyticum serovars 1, 3, 6, and 14 of biovar 1 which could be used as the basis of a practical molecular-serotyping method.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The reference strains used
were U. urealyticum serovar 1 (ATCC 27813), serovar 2 (ATCC
27814), serovar 3 (ATCC 27815), serovar 4 (ATCC 27816), serovar 5 (ATCC
27817), serovar 6 (ATCC 27818), serovar 7 (ATCC 27819), serovar 8 (ATCC
27618), serovar 9 (ATCC 33175), serovar 10 (ATCC 33699), serovar 11 (ATCC 33695), serovar 12 (ATCC 33696), serovar 13 (ATCC 33698), and
serovar 14 (ATCC 33697). Clinical isolates of serovars 1 to 14, which
had been serotyped by conventional methods, were kindly provided by
H. L. Watson (Department of Microbiology, University of Alabama at Birmingham, Birmingham) Escherichia coli DH5
was the host
for plasmid pUC19, which was obtained from the Institute of Genetics, Chinese Academy of Science.
Clinical specimens.
Cervical swabs from 200 female sex
workers and 100 other female clients attending the Sexually
Transmissible Diseases (STD) Clinic and 100 pregnant women attending
the Antenatal Clinic of the First Hospital of Beijing Medical
University were collected to test the practical usefulness of the
direct detection, biotyping, and serotyping methods. DNA was prepared
for PCR (see below) as soon as possible after receipt of specimens in
the laboratory. Cultures were not performed for these specimens.
Oligonucleotide primers.
Oligonucleotide primers UMS-125,
UMA226, UMS51, and UMA427, based on the previously published sequence
of the serovar 3 MBA gene (19, 22), were used for
amplification of the 5' ends of the U. urealyticum serovar
1, 3, 6, and 14 MBA genes. UMS-125 (GTA TTT GCA ATC TTT ATA TGT TTT CG)
and UMA226 (CAG CTG ATG TAA GTG CAG CAT TAA ATT C) were used without
modification. UMS51 and UMA427 were modified as follows. (i) Primers
UMS51 and UMA427 were modified by adding BamHI and
EcoRI restriction enzyme sites, respectively (underlined in
the sequences below), to facilitate cloning of the coding DNA sequences
of the 5' ends of the serovar 1, 3, and 14 MBA genes. (ii) In
ureaplasmas, the sequence TGA encodes tryptophan (amino acid 13), but
in E. coli it represents a stop codon. Therefore, a point
mutation was introduced into UMS51 (at base 38) to change the sequence
to TGG, which also encodes tryptophan. The modified primer sequences
used were UMS51, TGT GGA TCC TTC TGG GCT ATG
ACA TTA GGT GTT ACC (BamHI site underlined) and UMA427, CTC
GAA TTC ACC TGG TTG TGT AGT TTC AAA GTT CAC
(EcoRI site underlined).
The additional primers UMA269 (serovar 3/14 specific), UMA269' (serovar
1 and 6 specific), and UMS-54 (serovar 6 specific) were designed, using
the sequence data generated by this study, and were used for PCR
identification (see below). The sequences of these primers are as
follows: UMA269 (serovar 3/14), CTA AAT GAC CTT TTT CAA GTG TAC;
UMA269' (serovars 1 and 6), CCA AAT GAC CTT TTG TAA CTA GAT; and UMS-54
(serovar 6), CTT AGT GTT CAT ATT TTT TAC TAG.
DNA preparations.
Cells from 0.5 ml of ureaplasma broth
(10B) cultures of each U. urealyticum serovar were harvested
in late logarithmic growth phase by centrifugation at 14,000 × g for 20 min; clinical specimens were processed directly. DNA
was isolated from both cultures and clinical specimens by treatment
with 500 µl of digestion buffer (10 mM Tris-HCl, pH 8.0, 0.45%
Triton X-100, and 0.45% Tween 20) and proteinase K (100 µg/ml) at
55°C for 1 h and then extraction with phenol-chloroform-isoamyl
alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). DNA was
precipitated with 1/10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The washed and dried pellets were hydrated in 200 µl of ultrapure sterile water.
PCR.
The 25-µl amplification reaction mixtures contained
2.5 µl of 10× PCR buffer (1× PCR buffer is 10 mM Tris-HCl [pH 8.8 at 25°C], 1.5 mM MgCl2, 50 mM KCl, and 0.1% Triton
X-100), 0.5 U of Taq polymerase (Finnzymes OY, Espoo,
Finland), 200 µM (each) deoxynucleoside triphosphate (dATP, dCTP,
dGTP, and dTTP) (Boehringer, Mannheim, Germany), 10 pmol of each
primer, 5 µl of sample DNA, and ultrapure sterile water added to 25 µl. In each reaction, positive and negative controls were processed
in parallel with the tested samples to detect possible inhibition or contamination.
The denaturation, annealing, and elongation temperatures and times used
were 95°C for 30 s, 58°C for 30 s, and 72°C for 1
min,
respectively, for 40 cycles with a Perkin-Elmer thermocycler.
Eight
microliters of PCR product was analyzed by electrophoresis
on 2.0%
agarose gels which were stained with 0.5 µg of ethidium
bromide/ml. A
visible band of the appropriate size on UV translumination
was
considered a positive
result.
Cloning and sequencing.
The amplified fragments of the 5'
ends of U. urealyticum MBA genes (amplified with
UMS-125-UMA226 for serovar 6 and UMS51-UMA427 for serovars 1, 3, and
14) were ligated to plasmid vector pUC19, which had been digested by
SmaI. The ligation products were transformed into E. coli DH5, and positive transformants were identified by PCR,
using the primers UMS51-UMA427 for serovars 1, 3, and 14 and
UMS-125-UMA226 for serovar 6. The positive clones were sequenced in
two directions with the ABI 373A DNA-sequencing system.
To confirm the results of cloning experiments, the PCR products of both
sets of primers for ATCC reference strains and clinical
isolates of all
four serovars, 1, 3, 6, and 14, were directly
sequenced in two
directions with the ABI 373A DNA-sequencing
system.
Identification of serovars by PCR with restriction enzyme
analysis and serovar-specific PCR.
Primers UMS-125, UMA226,
UMA269, and UMA269' were used to amplify DNA from reference (ATCC)
strains, from clinical isolates that had been serotyped previously by
conventional methods, and directly from clinical specimens. Primers
UMS-125 and UMA226 were used to detect and biotype U. urealyticum. Primers UMS-125 and UMA269 were specific for serovars
3/14; primers UMS-125 and UMA269' were specific for serovars 1 and 6. Two different methods were used to further identify U. urealyticum serovars 1 and 6 (Fig. 1).
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FIG. 1.
Algorithm for identification of serovars of U. urealyticum biovar 1 in clinical specimens or cultures. (A)
Biotyping of U. urealyticum; (B) serotyping of U. urealyticum biovar 1.
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(i) Restriction enzyme analysis.
Ten microliters of the PCR
product of primers UMS-125 and UMA269' was treated with the restriction
enzyme HinfI (New England Biolabs, Beverly, Mass.) and
analyzed by electrophoresis. Those that were cut by the enzyme were
identified as serovar 1, those not cut were identified as serovar 6, and those that were partially digested were assumed to be mixed
serovars 1 and 6.
(ii) Confirmatory PCR for serovar 6.
Primers UMS-54
(specific for serovar 6) and UMA269' were used in a separate PCR to
confirm the identity of serovar 6-positive isolates or specimens.
Nucleotide sequence accession numbers.
The sequence data
obtained have been accepted by the GenBank nucleotide sequence database
and have been assigned accession no. AF056982 (serovar 14), AF056983
(serovar 1), and AF056984 (serovar 6). They will also appear in EMBL in
Europe and the DNA database Bank of Japan.
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RESULTS |
PCR.
The amplified fragments of the 5' ends of the MBA genes
of all 14 U. urealyticum serovars obtained by PCR with
primers UMS-125 and UMA226 were 403 bp (404 bp for serovar 6) for
biovar 1 and 448 bp for biovar 2; those obtained with primers UMS51 and
UMA427 were 447 bp for biovar 1 only. Both primer sets allowed a clear distinction between biovar 1 (serovars 1, 3, 6, and 14) and biovar 2 (serovars 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13).
Cloning and sequencing.
At least two positive clones were
identified for each of the amplified fragments of the 5' ends of the
MBA genes of all four serovars of U. urealyticum biovar 1, and at least one was chosen for sequencing. The other amplified
fragments were directly sequenced from two directions. The sequences of
amplicons from clinical isolates of serovars 1, 6, and 14 were
identical to those of the ATCC reference strains.
Comparison of the 5' end sequences of MBA genes and differentiation
among serovars by PCR and restriction enzyme analysis.
The
sequences of the MBA genes of U. urealyticum serovars 1, 3, 6, and 14 were compared with the published sequence for serovar 3 by
using multiple sequence alignment, as shown in Fig.
2. The primer sequences and the
restriction sites for relevant enzymes are shown. The sequence of the
5' end of the U. urealyticum serovar 3 MBA gene was
identical with the previously published sequence (22). The
sequence of the serovar 14 MBA gene was different from that of serovar
3 at only three sites. Differences between the sequences of the 5' ends
of the serovar 3, 14, 1, and 6 MBA genes occurred at 37 sites, as shown
in Table 1.
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FIG. 2.
Multiple sequence alignment of the 5' ends of MBA gene
sequences of U. urealyticum serovars 1, 3, 6, and 14 (ATCC
strains). Relevant primers (arrows) and restriction enzyme sites (bars)
are shown.
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TABLE 1.
Comparative analysis of the 5' end sequences of MBA genes
among four serovars of U. urealyticum biovar 1
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Individual differences that could be used for restriction enzyme
analysis were identified. Serovar 6 differed from serovar
1 and from
serovars 3/14 in having a base change from T to C at
position
6, with
loss of the
HinfI restriction enzyme site. This
difference
was used to distinguish serovars 6 and 1 after amplification
with
primers UMS-125 and UMA269' and treatment with
HinfI. The
PCR products of serovar 1, but not those of serovar 6, were cut
by
HinfI (Fig.
3).
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FIG. 3.
Results of PCR amplification of MBA genes with primers
UMS-125 and UMA269' followed by restriction enzyme analysis with
HinfI. Lanes M, molecular weight markers  X174
DNA/HaeIII; lanes 1 and 2, U. urealyticum serovar
1 ATCC strain PCR products before and after digestion by
HinfI; lanes 3 and 4, U. urealyticum serovar 1 clinical isolate PCR products before and after digestion by
HinfI; lanes 5 and 6, U. urealyticum serovar 1 positive clinical specimen PCR products after digestion by
HinfI; lanes 7 and 8, U. urealyticum serovar 6 ATCC strain PCR product before and after digestion by HinfI;
lane 9, U. urealyticum serovar 6 clinical isolate PCR
product after digestion by HinfI; lanes 10 to 13, PCR
products of four clinical specimens digested by HinfI. Lanes
10 and 12 were identified as serovar 1, and lanes 11 and 13 were
identified as serovar 6.
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The 5' ends of the MBA gene sequences of serovars 1 and 6 differed from
those of serovars 3/14 at two restriction endonuclease
sites. (i) At
base 251, C in serovars 3/14 is replaced by T in
serovars 1 and 6, with
loss of the
PvuII restriction enzyme site.
DNA from serovars
1, 3, 6, and 14 was amplified with primers UMS51
and UMA427, and the
products were compared before and after digestion
with
PvuII. The PCR products of serovars 3 and 14, but not those
of serovars 1 and 6, were cut with
PvuII. (ii) At bases 269 to
272, GTAC in serotypes 3/14 is replaced by ATCT in serovars 1
and 6, with loss of the
RsaI restriction enzyme site. The presence
of several differences in the sequences between bases 269 and
282 allowed us to design new primers, UMA269 and UMA269'; which
were
specific for serovars 3/14 and serovars 1 and 6, respectively,
and
which could be used with UMS-125 to distinguish these two
serovar pairs
by PCR. The sequence differences at
54,
55, and
56 also allowed
us to design primer UMS-54, which was specific
for serovar 6 and can be
used with UMA269' to differentiate serovar
6 from serovar
1.
The specificity of primers UMA269-UMA269' and UMS-54 were tested with
all 14 serovars of
U. urealyticum, and the results are
shown
in Fig.
4,
5, and
6.
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FIG. 4.
Results of PCR amplification of MBA genes of all 14 serovars of U. urealyticum with primers UMS-125 and UMA269
(specific for serovars 3/14). A positive result is shown by a 442-bp
band. Lanes M, molecular weight markers X174 DNA/HaeIII;
lanes 1 and 2, U. urealyticum serovars 1 and 2 ATCC strains;
lanes 3 and 4, U. urealyticum serovar 3 ATCC strain and
clinical isolate; lanes 5 to 14, U. urealyticum serovars 4 to 13 ATCC strains; lanes 15 and 16, U. urealyticum serovar
14 ATCC strain and clinical isolate.
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FIG. 5.
Results of PCR amplification of MBA genes of all 14 serovars of U. urealyticum with primers UMS-125 and UMA269'
(specific for serovars 1 and 6). A positive result is shown by a 442- or 443-bp band. Lanes M, molecular weight markers X174
DNA/HaeIII; lanes 1 and 2, U. urealyticum serovar
1 ATCC strain and clinical isolate; lanes 3 to 6, U. urealyticum serovars 2 to 5 ATCC strains; lanes 7 and 8, U. urealyticum serovar 6 ATCC strain and clinical isolate; lanes 9 to
16, U. urealyticum serovars 7 to 14 ATCC strains.
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FIG. 6.
Results of PCR amplification of MBA genes of all 14 serovars of U. urealyticum with primers UMS-54 (specific for
serovar 6) and UMA269'. A positive result is shown by a 369-bp band.
Lanes M, molecular weight markers X174 DNA/HinfI; lanes 1 and 2, U. urealyticum serovar 1 ATCC strain and clinical
isolate; lanes 3 to 6, U. urealyticum serovars 2 to 5 ATCC
strains; lanes 7 and 8, U. urealyticum serovar 6 ATCC strain
and clinical isolate; lanes 9 to 16, U. urealyticum serovars
7 to 14 ATCC strains.
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Detection, biotyping and serotyping of U. urealyticum
biovar 1 from clinical specimens.
The results of direct PCR for
detection and biotyping of U. urealyticum in clinical
specimens are shown in Table 2. The
U. urealyticum MBA gene was identified by PCR in 185 (46.3%) of 400 cervical swabs. Serovars belonging to biovar 1 only
were detected in 151 swabs (37.8%), biovar 2 strains only were
detected in 20 swabs (5.0%), and strains belonging to both biovars
were detected in 14 swabs (3.5%). U. urealyticum was
detected significantly more frequently in cervical swabs of pregnant
women (58.0%) than in those of sex workers and other women attending
the STD clinic (42.3%) (P < 0.01; odds ratio, 1.9;
95% confidence interval, 1.2 to 3.0).
The serovars identified in 165 cervical swabs in which
U. urealyticum biovar 1 was detected are shown in Table
3. Serovar
1 and serovars 3/14 were found
most commonly (and in approximately
equal numbers of specimens) in 77 (46.7%) and 76 (46.1%) specimens,
respectively; serovar 6 was found
in 42 (25.5%) positive specimens.
A high proportion of specimens
(22.2% overall) contained more
than one serovar.
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DISCUSSION |
The two biovars of U. urealyticum can be distinguished
by various molecular techniques (5, 13, 14, 18), while
serotyping by conventional methods remains difficult. Currently, there
are no commercially available antisera, and cross-reactions were common even when monoclonal antisera were used (4, 9, 12). Faster and more specific serotyping methods are required to confirm the reported associations of particular serovars with diseases in which
U. urealyticum has been implicated.
In this study we amplified the 5' ends of the MBA genes of the four
U. urealyticum serovars belonging to biovar 1. The amplified DNA was cloned into E. coli by using the plasmid pUC19. The
clones were used to provide DNA for sequencing and have been used to express biovar-specific MBA genes for use in serological tests to
measure biovar-specific antibody responses of patients with suspected
U. urealyticum infections (to be published separately). We
have shown that the 5' ends of the MBA genes in serovars 3 and 14 are
very similar, with base differences occurring only at three sites. We
have demonstrated significant differences between serovars 3/14, 1, and
6 of biovar 1 and, based on these differences, we have used a
combination of biovar-specific PCR, restriction enzyme analysis, and
serovar-specific PCR to identify individual serovars of biovar 1. This
allows rapid biotyping and serotyping of isolates and direct detection
and serovar identification of U. urealyticum in clinical
specimens, the majority of which belong to biovar 1.
To test the practical usefulness of our methods, genital specimens from
three groups of women were tested. U. urealyticum was
detected in nearly half (46.3%) of the cervical swabs tested, and of
these, 89.2% contained biovar 1 and 18.3% contained biovar 2. Serovars 3/14 and 1 were each identified, alone or with other serovars,
in nearly half of the specimens containing biovar 1, 22.2% of which
contained more than one serovar. Pregnant women were significantly more
likely to be colonized with U. urealyticum, and
specifically, with serovar 1. This appeared to be attributable to a
higher proportion of pregnant women in whom serovars 3/14 alone were
found. Otherwise, there appeared to be no difference in the
distribution of serovars between the different groups of subjects. The
reason for this difference is uncertain, but it could be due to
hormonal effects, which could increase ureaplasma counts, and thus the
likelihood of detection, during pregnancy. The distribution of serovars
is consistent with that found in other studies in which U. urealyticum isolates have been serotyped, using
indirect-immunofluorescence tests, by antisera or monoclonal antibodies
(4, 9, 23). For example, Naessens et al. showed that more
than 90% of colonized women had isolates belonging to biovar 1, of
which 52.2% were serovar 3, 30.3% were serovar 6, and 9.5% were
serovar 1 (9).
Further investigation is needed to distinguish serovar 3 from serovar
14. It has recently been shown that there are differences in the 3'
ends of the MBA gene repetitive units of these serovars, demonstrating
potential alternative sites of difference. In the future, biotyping and
serotyping by direct use of biovar-specific and even serovar-specific
primers will assist in the study of the pathogenesis and epidemiology
of U. urealyticum infections, which until now has been
restricted by the practical difficulties of conventional methods.
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ACKNOWLEDGMENTS |
We thank Xueqian Gong and Shouyi Chen for their precious help in
cloning and sequencing and Greg James, Peter Jelfs, and Zhenfang Ma for
their assistance with this project.
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FOOTNOTES |
*
Corresponding author. Mailing address: Centre for
Infectious Diseases and Microbiology, Institute of Clinical Pathology
and Medical Research, Westmead Hospital, Darcy Rd., Westmead, New South
Wales, 2145 Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: lyng{at}cidm.wsahs.nsw.gov.au.
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Journal of Clinical Microbiology, March 1999, p. 538-543, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
