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Journal of Clinical Microbiology, November 2001, p. 3871-3876, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3871-3876.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of a p28 Gene in Ehrlichia
ewingii: Evaluation of Gene for Use as a Target for a
Species-Specific PCR Diagnostic Assay
Asiya A.
Gusa,1
Richard S.
Buller,2
Gregory A.
Storch,2
Mark M.
Huycke,3,4
Linda J.
Machado,3,4
Leonard N.
Slater,3,4
Steven L.
Stockham,5 and
Robert F.
Massung1,*
Division of Viral and Rickettsial Diseases, National Center
for Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia1; The Edward
Mallinckrodt Department of Pediatrics, Washington University School of
Medicine and St. Louis Children's Hospital, St.
Louis,2 and Department of Veterinary
Pathobiology, College of Veterinary Medicine, University of
Missouri
Columbia, Columbia,5 Missouri; and
Division of Infectious Diseases, Department of Medicine,
University of Oklahoma Health Sciences Center,3
and Department of Veteran's Affairs Medical
Center,4 Oklahoma City, Oklahoma
Received 8 May 2001/Returned for modification 3 July 2001/Accepted 12 August 2001
 |
ABSTRACT |
PCR was used to amplify a 537-bp region of an Ehrlichia
ewingii gene encoding a homologue of the 28-kDa major antigenic
protein (P28) of Ehrlichia chaffeensis. The E.
ewingii p28 gene homologue was amplified from DNA extracted
from whole blood obtained from four humans and one canine with
confirmed cases of infection. Sequencing of the PCR products (505 bp)
revealed a partial gene with homology to outer membrane protein genes
from Ehrlichia and Cowdria spp.:
p30 of Ehrlichia canis (
71.3%),
p28 of E. chaffeensis (68.3%), and
map1 of Cowdria ruminantium (67.3%). The
peptide sequence of the E. ewingii partial gene product
was deduced (168 amino acids) and the antigenicity profile was
analyzed, revealing a hydrophilic protein with 69.1% identity to P28
of E. chaffeensis, 67.3% identity to P30 of E.
canis, and 63.1% identity to MAP1 of C.
ruminantium. Primers were selected from the E. ewingii
p28 sequence and used to develop a species-specific PCR
diagnostic assay. The p28 PCR assay amplified the
expected 215-bp product from DNA that was extracted from EDTA-treated
blood from each of the confirmed E. ewingii infections
that were available. The assay did not produce PCR products with DNA
extracted from E. chaffeensis-, E.
canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28 assay for
E. ewingii. The sensitivity of the E.
ewingii-specific PCR assay was evaluated and determined to
detect as few as 38 copies of the p28 gene.
| |
INTRODUCTION |
Human ehrlichioses are
emerging tick-borne infections that were first described in 1987 (12). Prior to 1999, the two known etiologic agents of
human ehrlichiosis in the United States were Ehrlichia
chaffeensis (1) and Ehrlichia
phagocytophila (commonly referred to as the agent of human
granulocytic ehrlichiosis) (7). In 1999, Ehrlichia
ewingii, a pathogen originally recognized to cause granulocytic
ehrlichiosis in canines (2), was implicated in causing
several cases of granulocytic ehrlichiosis in humans (4).
Since the earliest case, retrospectively identified from 1996, approximately 10 confirmed cases of granulocytic ehrlichiosis caused by
E. ewingii have been identified from Missouri
(4) and Oklahoma (C. D. Paddock, personal
communication). Patients infected with E. ewingii experience
symptoms similar to those of other human ehrlichioses, such as fever,
headache, and myalgia, and usually report recent exposure to ticks.
Many of the humans infected by E. ewingii were receiving
immunosuppressive therapy at the time of infection (4).
Based on the 16S rRNA sequence, E. ewingii is most closely
related to E. chaffeensis (98.1% homology) and
Ehrlichia canis (98.0% homology), the agent of canine
monocytic ehrlichiosis (2). Of these three, E. chaffeensis has been studied in the most detail. DNA sequences are
available for numerous E. chaffeensis genes, including those
encoding 16S rRNA, groESL, p120 antigen, the variable-length PCR
target, and the p28 gene family (1, 17-19, 22, 23,
27, 28). The p28 gene family represents a series of
21 homologous genes (20 to 83% amino acid identity) that are arranged
in tandem in the E. chaffeensis genome
(28). The p28 genes encode major antigenic proteins of E. chaffeensis as
determined by Western blotting (5, 6, 20). Recombinant
forms of the p28 genes and the corresponding homologous
p30 genes of E. canis have been expressed and
shown to be useful for the serodiagnosis of these agents by use of
Western and dot blot immunoassays (16, 25, 26). It has
been suggested that these genes may play a role in immune response
evasion through differential expression of the multigene locus
(14, 18, 19). At least 6 of the 21 E. chaffeensis
p28 genes and five of five E. canis p30 genes were transcriptionally active when examined in cell culture (15, 28). Two homologues of the p28 gene family have also
been described for Ehrlichia muris, another member of the
E. canis genogroup (GenBank accession numbers AF165813 and
AF165814).
The diagnostic assays currently available for the identification of
infection by E. ewingii have limitations. The most reliable test for identifying an ehrlichial infection is isolation of the agent
in tissue culture (8). However, E. ewingii has
not yet been cultured in vitro. Indirect fluorescence antibody assays are commonly used for the diagnosis of E. chaffeensis and
E. canis infections, and while either of these may
serve as a surrogate antigen to screen for suspected E. ewingii infections, an indirect fluorescence antibody assay using
surrogate antigens cannot be confirmatory (9, 12).
Laboratory confirmation of E. ewingii infection has
ultimately been determined by molecular methods, particularly PCR. In
contrast to the numerous genes that have been identified and
sequenced from the E. chaffeensis genome, only
two genes, the 16S rRNA and groE genes, have been
characterized and may currently serve as PCR targets for E. ewingii (3, 4, 24). This study describes the
identification and characterization of a gene in E. ewingii
that is homologous to the major antigenic proteins of E. chaffeensis (p28) and E. canis
(p30). The unique regions of the E. ewingii p28
homologue were used to design primers for a species-specific PCR
diagnostic assay. The ability of the p28 gene PCR assay to
identify and differentiate infection by E. ewingii is also
evaluated in this study.
| |
MATERIALS AND METHODS |
Samples and DNA extraction.
DNA was extracted from each
sample in this study using the QIAamp blood kit (Qiagen Inc., Valencia,
Calif.). Control samples included DNA extracted from EDTA-treated human
blood specimens that were previously confirmed at the Centers for
Disease Control and Prevention as E. chaffeensis infected,
and DNA was extracted from tissue cultures infected with either
E. canis strain Arkansas (DH82 cells) or E. phagocytophila strain USG3 (HL60 cells).
PCR amplification.
PCR amplifications were done in a 9600 thermal cycler (Perkin-Elmer, Applied Biosystems, Foster City,
Calif.) using the Taq Master PCR kit (Qiagen). The
final volume for each reaction was 25 µl, with reagent concentrations
of 0.5 µM for each primer, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM
MgCl2, 200 µM for each deoxynucleoside triphosphate, 1.5 U of Taq DNA polymerase, and 2.5 µl of
template. All PCRs used the following conditions: 95°C (2 min)
followed by 40 cycles of 94°C (30 s), variable annealing temperature
(30 s), and 72°C (1 min), followed by an extension period (72°C, 5 min).
Primers FECH2 (5'-ACATCAGTGGAAAATACATG) and REC1
(5'-ACCTAACTTTCCTTGGTAAG) were derived from primers used in
a previous study (19) to amplify the p28 of
E. chaffeensis. These primers were initially used to amplify
the p28 gene from the human and canine E. ewingii-infected samples, using an annealing temperature of 45.7°C. The PCR diagnostic assay targeting the p28 gene of
E. ewingii used primers EEM2F
(5'-GGAGCTAAAATAGAAGATAATC) and EEM1R (5'-GTGCCAAAAGGT
AATACAT) with an annealing temperature of 55°C.
The annealing temperatures for each PCR assay were optimized by
use of a temperature gradient thermal cycler (Mastercycler
gradient;
Eppendorf Scientific, Westbury, N.Y.). Results of the
PCRs were
assessed by electrophoresis of 6 µl of each product
in a 2% agarose
gel containing ethidium
bromide.
Cloning of pMAP.
Amplified product from the FECH2-REC1 PCR
of E. ewingii OK-1 DNA was cloned into vector pCR 2.1 using
the Original TA cloning kit (Invitrogen, Carlsbad, Calif.). The
presence of the insert was confirmed by DNA sequencing. This plasmid,
named pMAP, was subsequently used as a positive control for evaluation
of the p28 assay.
Sensitivity assessment.
The sensitivity of the assay was
determined, using the vector pMAP, through a previously developed
method (13). In brief, 10-fold serial dilutions of a known
quantity of pMAP were added to aliquots of DNA extracted from
uninfected EDTA-treated human blood. The assay was done on the dilution
series, and the limit of detection was determined by the final dilution
at which a PCR product was still visible. The dilution series was
quantitated using an MBA 2000 spectrophotometer (Perkin-Elmer, Norwalk,
Conn.).
Nucleotide sequencing.
PCR products were purified using a
Wizard PCR Preps DNA Purification Kit (Promega, Madison, Wis.). The
purified PCR products were sequenced with forward and reverse primers
using the Prism Ready Reaction DyeDeoxy Cycle Sequencing kit in a 9600 Perkin-Elmer thermal cycler (Perkin-Elmer, Applied Biosystems).
Unincorporated fluorescence-labeled dideoxynucleoside triphosphates
were removed using the Dye-Ex Spin kit (Qiagen). Sequencing reaction
products were separated and sequence data collected using a 377 ABI
automated sequencer (Perkin-Elmer, Applied Biosystems).
Data analysis.
Nucleotide sequences were edited and
assembled using the STADEN sequence analysis package (10).
Sequence homology comparisons and multiple sequence alignments were
made with the GAP and PILEUP programs, respectively, of the Genetics
Computer Group (Madison, Wis.) package (11). Nucleotide
sequence homology searches were made through the National Center for
Biotechnology Information BLAST network service. Phylogenetic analysis
was done by use of the PAUP program (version 4.0.0d64) on a Power
Macintosh 9500/132.
| |
RESULTS |
Analysis of an E. ewingii p28 homologue.
Four
human samples and one canine sample infected with E. ewingii
were obtained (Table 1) that had been
confirmed infected at the Centers for Disease Control and Prevention by
PCR amplification of the 16S rRNA gene and/or the groESL
gene. PCR products were obtained from each of the five E. ewingii-infected samples using primers specific for a partial gene
fragment of a p28 of E. chaffeensis, the
Arkansas strain (19). The sample designation (C-1, OK-1, OK-2, MO-1, or MO-3), sample source, location of collection, and GenBank accession number that was assigned to each E. ewingii p28 gene sequence are indicated in Table 1. Sequence analysis of
each product revealed a 505-bp region (Fig.
1) from which a 168-amino-acid peptide
could be deduced (Fig. 2). The sequence was highly conserved among the five samples; C-1 and OK-2 nucleotide and peptide sequences for this region were identical. The OK-1 and MO-3
sequences were also identical; however, they differed by two
nucleotides when compared with the C-1 and OK-2 sequences. This
nucleotide difference results in a peptide difference of only one amino
acid (position 153 in Fig. 2). The MO-1 sequence for this region was
the most divergent, differing by 19 nucleotides when compared to the
C-1 and OK-1 sequences, resulting in a protein coding difference of 11 amino acids. Database sequence homology searches of the 505-bp region
revealed nucleotide and peptide similarity of 70% to major outer
membrane protein homologues of species closely related to E. ewingii, namely, the p28 genes of E. chaffeensis, the p30 genes of E. canis, and
the map1 gene of Cowdria ruminantium. The
nucleotide and peptide sequence homologies of the E. ewingii
gene to the closest p28 homologues are shown in Table
2.
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FIG. 1.
Comparison of E. chaffeensis (E.
chaf.) p28-19 gene (Arkansas
strain; accession no. U72291) to the E. ewingii p28
genes (C-1, OK-1, OK-2, MO-1, and MO-3). Dots represent sequence
agreement, letters represent nucleotide differences, and the tilde
symbol indicates that the sequence was not available. All primers used
in this study are indicated by arrows for orientation.
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FIG. 2.
Deduced amino acid sequences of E.
ewingii P28 (C-1, OK-1, OK-2, MO-1, and MO-3) compared with the
amino acid sequences of the most-homologous copies of E.
chaffeensis (E. chaf.) P28 (Arkansas P28-19;
accession no. U72291), E. canis P30 (Oklahoma; accession
no. AF078553), and C. ruminantium MAP1 (Nyatsanga;
accession no. U50834).
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Based on the relative size of the complete gene sequences of the
E. chaffeensis and
E. canis major outer membrane
protein
homologues, the 505-bp region of the
p28 of
E. ewingii is predicted
to encode approximately 60% of the total
protein. This partial
protein sequence possesses numerous differences
that are unique
to
E. ewingii when compared with its closest
P28/P30 homologues,
with two particularly notable regions with
extensive differences
(Fig.
2). As shown in Fig.
2, there is a
6-amino-acid deletion
present in the
E. chaffeensis P28
protein, relative to the
E. canis P30 protein, that may
contribute to the apparent difference
in the molecular mass between the
E. chaffeensis (28-kDa) and
E. canis
(30-kDa) homologues. This deletion is also found in the
E. ewingii sequence, suggesting that the mature
E. ewingii
protein
may be similar in size to the
E. chaffeensis 28-kDa
homologue.
The amino acid composition of the
E. ewingii
partial protein revealed
a primarily hydrophilic protein, consistent
with P28 being a strongly
immunogenic protein (data not
shown).
A phylogenetic analysis was also used to compare the amino acid
sequence deduced from the copy of the
E. ewingii p28 gene
that was amplified from each of the four human and one canine
samples
to each other and to the 21 copies of the P28 protein
encoded in the
E chaffeensis genome (data not shown). This analysis
confirmed that the
E. ewingii-encoded P28 proteins comprise
a
very closely related group that is distinct from the
E. chaffeensis P28 proteins. Among the
E. chaffeensis P28
proteins, those encoded
by the
p28 genes
p28-
15 through
p28-
19
represented the closest
homologues to the
E. ewingii P28
proteins.
Development and evaluation of the E.
ewingii-specific PCR assay.
The nucleotide differences
between the p28 gene of E. ewingii and the
E. chaffeensis and E. canis homologues were
considered significant enough to utilize the gene sequence as a target
for a species-specific PCR assay. Primers were chosen in regions of the
gene fragment unique to E. ewingii. After testing numerous primer sets, primers EEM2F and EEM1R (shown in Fig. 1) amplified the
p28 gene of E. ewingii most efficiently and with
minimal background. The assay using these primers was tested on DNA
extracted from the five PCR-positive E. ewingii-infected
samples and from samples containing E. chaffeensis, E. canis, and E. phagocytophila (Fig. 3). The assay amplified a 215-bp region
of the p28 gene from DNA from each of the E. ewingii-infected samples (Fig. 3, lanes 1 to 5). Sequencing of the
PCR products confirmed amplification of a p28 gene of
E. ewingii. No products were obtained when the template
for the PCR assay was representative DNA from E. chaffeensis (Fig. 3, lanes 6 to 10), E. canis (Fig. 3, lane 11), and
E. phagocytophila (Fig. 3, lane 12).
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FIG. 3.
Species-specific p28 PCR assay results
for E. ewingii with primers EEM2F and EEM1R. The first
five lanes show PCR products obtained with the E.
ewingii-infected template DNAs (lane 1, C-1; lane 2, OK-1; lane
3, OK-2; lane 4, MO-1; and lane 5, MO-3). The remaining lanes show PCR
products amplified from DNAs from samples containing E.
chaffeensis (lanes 6 to 10), E. canis (lane 11),
and E. phagocytophila (lane 12) as well as
positive (+) and negative ( ) controls. The expected size (215 bp) is
indicated. Lanes M show size standards (HaeIII digest of
phage  X174 DNA).
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Sensitivity of the E. ewingii-specific PCR
assay.
The sensitivity of the E. ewingii assay was
determined by performing a PCR amplification using a dilution series of
a known amount of a plasmid containing the E. ewingii p28
gene (pMAP) in the presence of DNA extracted from uninfected
EDTA-treated human blood. Results of the sensitivity testing are shown
in Fig. 4. The assay detected as few as
38 copies of the p28 gene (Fig. 4, lane 7).
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FIG. 4.
Sensitivity of the species-specific p28
PCR assay for E. ewingii with primers EEM2F and EEM1R. A
10-fold dilution series of pMAP template amplified by the
p28 PCR assay is shown (lane 1, 3.84 × 107 copies of pMAP, through lane 8, 3.84 copies of pMAP).
The expected size (215 bp) is noted. Lanes M show size standards
(HaeIII digest of phage X174 DNA).
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DISCUSSION |
Molecular and serologic evidence suggests that E. ewingii is closely related to two other members of the
Ehrlichia genus, E. chaffeensis and E. canis. Whereas multigene families encoding homologous 28- to
30-kDa immunodominant outer membrane proteins have been described for
E. chaffeensis and E. canis, no such
homologue had been identified previously for E. ewingii. Previous studies using sera from E. ewingii-infected human and canine samples either did not react to
native and recombinant 28-kDa (P28) and 30-kDa (P30) protein antigens
of E. chaffeensis and E. canis (4,
25) or showed weak reactivity (20). This suggested
that if E. ewingii does encode P28/P30 homologues these
proteins would exhibit significantly different antigenic properties
compared to the homologues in E. chaffeensis and
E. canis.
In this report we describe the PCR amplification and cloning of a
partial open reading frame from E. ewingii that encodes a
member of the p28/p30 gene family. The fact that
the E. ewingii P28 protein shows only 70% homology to the
corresponding antigens of E. chaffeensis and E. canis may explain the absence of seroreactivity to this range of
antigens in patients infected by E. ewingii. The
p28 gene described in this report is likely to be the first of numerous homologous genes to be described in E. ewingii. Loci encoding multiple copies of the homologous
p28, p30, and map1 genes have been
described for E. chaffeensis, E. canis, and
C. ruminantium, respectively, and similar multigene loci are
likely to be found in the E. ewingii genome (14-19,
28). Two partial copies of p28 genes of E
muris were deposited recently in GenBank (accession numbers
AF165813 and AF165814). Like the E. chaffeensis and E. canis homologues, the E. muris genes showed 72 to 76%
identity over a 302-bp region to the E. ewingii p28 genes
(data not shown). Of the p28 copies that have been described
in E. chaffeensis, the E. ewingii p28 gene
identified in this paper has the highest nucleotide homology to
p28-18, previously referred to as
omp-1F (17) and
orf-4 (19), and
p28-19, previously referred to as p28
(17) and orf-5 (19). Both
p28-18 and p28-19 have been shown to be transcriptionally active (19, 28).
Phylogenetic analysis of the amino acid sequences predicted from the
E. ewingii p28 genes shows them to be most closely related
to the P28 proteins encoded within the p28-15 to
p28-19 gene cluster (28). On the basis of the relatively high homology of these five genes to the E. canis and E. muris p28/p30
homologues, Yu et al. (28) have suggested that these genes
represent the precursors of the current 21 copies that comprise the
E. chaffeensis p28 gene family, and our findings support
this hypothesis (17, 28). Of interest, the P23 antigen
that is expressed in E. chaffeensis is believed to be
derived from the p28-18 open reading frame
following posttranslational cleavage (17). Although we
have no evidence that the E. ewingii p28 gene is expressed,
on the basis of the antigenicity profile of the protein, it is likely
that it will be an immunodominant antigen and a suitable candidate for
serodiagnosis. These questions will be more readily answered once the
organism is cultured and expression studies can be performed.
Whereas the P28 protein sequences for four of the E. ewingii
samples (C-1, OK-1, OK-2, and MO-3) were highly homologous (>99.4% identity), the P28 sequence from sample MO-1 showed 94.0 to 94.6% identity to each of the others. There are several possible explanations for the difference between the MO-1 P28 sequences and the others. It is
possible that a different copy of the p28 gene was amplified from the MO-1 sample than was amplified from each of the other samples.
Alternatively, it may be that the MO-1 sample represents a different
strain of E. ewingii, although the 16S rRNA gene sequences were identical for MO-1 and the other samples. The latter hypothesis is
supported by the low homology noted for the E. chaffeensis p28 gene family, for which each copy of the P28 protein shows <83% identity to the other copies (28). Similarly low
P28 protein homology has been shown among the multiple genes identified
in E. canis (five copies; <74% identity), E. muris (two copies; <81% identity), and C. ruminantium
(two copies; <47% identity) (15, 21). Although these
data suggest that we have amplified the same copy of the E. ewingii p28 gene from each of the five samples tested, the
examination of additional genes, particularly single-copy genes that
are more variable than the 16S rRNA, are needed to determine whether
MO-1 represents a variant strain.
The proper diagnosis of infection by E. ewingii is extremely
important in determining the incidence and prevalence of infection and,
in particular, whether these numbers are increasing. However, the
limited number of genes that have been identified for E. ewingii and the close antigenic relationship of E. ewingii to E. chaffeensis and E. canis have
restricted the molecular and serologic tools available for the
diagnosis of human E. ewingii infections. There currently
are no serologic assays designed to specifically identify infection by E. ewingii. In fact, previous serologic assays
may have incorrectly identified E. ewingii infections
as infections with E. chaffeensis due to the serologic
cross-reactivity of these two agents (4). Therefore,
molecular-based assays provide the most specific and sensitive means of
identifying E. ewingii infections. Alignment of the E. ewingii P28 protein with the P28/P30 homologues from other species
of Ehrlichia revealed two variable regions with numerous
amino acid differences from these proteins (Fig. 2). These two regions
were previously described as being hypervariable for the
p28/p30 genes of E. chaffeensis and
E. canis and the map1 genes of C.
ruminantium (15, 17, 19, 21, 28). These hypervariable
regions were used to develop a PCR assay that targets the
p28 of E. ewingii and provides a useful
species-specific diagnostic tool for molecular confirmation of
infection by E. ewingii. The assay successfully identified
each of five cases of confirmed infection by E. ewingii and
did not amplify DNA from closely related species (E. chaffeensis, E. canis, and E. phagocytophila). In addition, the sensitivity of the
assay was shown by the detection of as few as 38 copies of the gene.
Identification of the p28 gene in E. ewingii
presents several possibilities for utilizing the protein in serologic
diagnostic assays. Serologic assays have a wider applicability due to
the more frequent availability of patient sera than whole blood and to
the lack of expensive equipment and expertise needed to perform PCR in
most clinical facilities. Because the protein has only a 70%
similarity to its nearest homologue, the P28 of E. ewingii may prove useful in species-specific identification in serologic assays. On the other hand, knowledge of the conserved antigenic regions
between the P28 and P30 homologues may lead to a broad-spectrum serologic assay for detection of ehrlichiosis caused by E. chaffeensis, E. canis, and E. ewingii.
| |
ACKNOWLEDGMENTS |
We are grateful to Chris Paddock for his contributions throughout
the study and to the CDC Biotechnology Core Facility for the synthesis
of oligonucleotides. We thank Greg Dasch and Jamie Childs for review of
the manuscript and useful suggestions.
This study was supported in part by the APHL-CDC Emerging Infectious
Diseases Fellowship Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centers for
Disease Control and Prevention, 1600 Clifton Rd., MS G-13, Atlanta, GA 30333. Phone: (404) 639-1082. Fax: (404) 639-4436. E-mail:
rfm2{at}cdc.gov.
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Journal of Clinical Microbiology, November 2001, p. 3871-3876, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3871-3876.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
