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Journal of Clinical Microbiology, October 1999, p. 3097-3101, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Semiquantitative Species-Specific Detection of Bartonella
henselae and Bartonella quintana by
PCR-Enzyme Immunoassay
Anna
Sander* and
Susanne
Penno
Abteilung Mikrobiologie und Hygiene, Institut
für Medizinische Mikrobiologie und Hygiene, Klinikum der
Universität Freiburg, Freiburg, Germany
Received 2 February 1999/Returned for modification 7 June
1999/Accepted 24 June 1999
 |
ABSTRACT |
Bartonella henselae is the main causative agent of
cat-scratch disease, and both B. henselae and
Bartonella quintana cause angioproliferative disorders such
as bacillary angiomatosis. To increase the sensitivity of
Bartonella detection by PCR and to improve the species
differentiation, we developed a semiquantitative, species-specific
PCR-based enzyme immunoassay (EIA). The 16S rRNA gene was selected as
the target sequence. Internal nucleotide sequences derived from the
amplified 16S rRNA region were used to develop species-specific
oligonucleotide probes for B. henselae and B. quintana. Biotin-labeled PCR products were immobilized on
streptavidin-coated microtiter plates, hybridized to a
digoxigenin-labeled probe, and detected with antidigoxigenin peroxidase
conjugate. No cross-hybridization with other Bartonella or
non-Bartonella species was observed. This EIA was as
sensitive as dot blot hybridization and was 10 times more sensitive
than visualization of PCR products on agarose gels. Serial dilutions of
B. henselae and B. quintana suspensions
demonstrated that an optical density (OD) of approximately 0.200 was
equivalent to 5 CFU in the reaction mixture. By comparing the OD of the
bacterial dilutions with that obtained from clinical specimens we could
determine that the number of CFU in clinical samples ranged from
103 to 106 CFU/ml. The PCR-EIA developed in the
present study is a rapid, sensitive, and simple method for the
diagnosis of B. henselae and B. quintana infections.
| |
INTRODUCTION |
The genus Bartonella
presently includes 14 species, but at present, only 5 of them are known
to be pathogenic for humans. B. bacilliformis is the agent
of bartonellosis, a diphasic illness which is endemic in the South
American Andes and which is limited to the valleys of Peru, Ecuador,
and Colombia (7). Only a few reports of human infections due
to B. elizabethae (8) and B. clarridgeiae (13, 15) have been published. The majority
of human infections are caused by the two species B. henselae, the main agent of cat-scratch disease (CSD), and
B. quintana, which causes trench fever and endocarditis,
most often observed in homeless, chronic alcoholic patients. CSD occurs
worldwide and is probably the most common Bartonella
infection. It is a common cause of subacute, regional lymphadenopathy
in mostly immunocompetent children and adults. Atypical manifestations
of CSD including Parinaud's oculoglandular syndrome, hepatic and
splenic abscesses, and central nervous system and pulmonary
manifestations are well characterized (2, 4).
Immunocompromised patients are more likely to have systemic infections
caused by both B. henselae and B. quintana.
Especially in human immunodeficiency virus-infected patients clinical
manifestations like bacillary angiomatosis (BA), bacillary peliosis
hepatis, osteolytic lesions, relapsing fever with bacteremia,
endocarditis, and encephalitis are common (2).
Laboratory methods for the diagnosis of Bartonella
infections include isolation of the organisms by culture, serological
assays, histopathological examination, and molecular detection of
Bartonella DNA in affected tissue (2, 21, 22).
However, Bartonella species are fastidious, slowly growing
bacteria, and routine bacterial culture protocols usually do not allow
detection of these organisms (19). Serological testing for
detection of antibodies to B. henselae in CSD patients seems
to be quite reliable when titers are 1:512 or higher. The
seroprevalence (usually low antibody titers) in healthy individuals is
high (up to 30%), and low antibody levels (between 1:64 and 1:256)
could indicate prior contact with B. henselae but could also
indicate the onset or the end of illness (22). Therefore,
the diagnosis of Bartonella infection in patients with low
antibody titers should be confirmed histologically and/or by detection
of Bartonella DNA in the affected tissue. Additionally, serological methods do not allow differentiation between B. henselae and B. quintana infections. The
cross-reactivity between these two species was demonstrated to be very
high (95%) in patients with CSD (22). Unfortunately, there
exist no reliable data concerning serological testing in
immunocompromised patients with Bartonella infections.
Recently, several PCR-based assays have been developed for detection of
Bartonella DNA in clinical specimens (1, 3, 5, 10, 16,
21, 23). Successful amplification of Bartonella DNA
depends not only on the primers used but also on the condition of the
specimens (native and non-formalin-fixed, frozen, or
formalin-fixed, paraffin-embedded specimens). Almost all primers used
for diagnosis of Bartonella infections are genus specific
but not species specific (1, 21). Therefore, hybridization
or sequencing of the amplified DNA is required for species
identification. Both methods are difficult, expensive, and
time-consuming.
The aim of the present study was to develop a rapid and simple method
for species-specific detection of amplified Bartonella DNA
by a PCR-based enzyme immunoassay (EIA).
| |
MATERIALS AND METHODS |
Bacterial strains.
B. henselae Houston-1 (ATCC 49882)
and Bartonella quintana CIP 103739 (Collection de
l'Institut Pasteur, Paris, France) were used as positive controls for
further testing. The strains were cultured on chocolate agar plates
containing 10% defibrinated sheep blood. The plates were incubated at
37°C in a 5% carbon dioxide atmosphere for 3 to 4 days. B. bacilliformis ATCC 35685, B. elizabethae ATCC 49927, B. clarridgeiae ATCC 51784, and the following
non-Bartonella species served as negative controls in the
present study: Afipia felis ATCC 53690, Staphylococcus
aureus, Streptococcus pyogenes, Streptococcus
agalactiae, Pseudomonas aeruginosa, Escherichia
coli, Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter diversus. The non-American
Type Culture Collection (ATCC) strains were clinical isolates of the
Institut für Medizinische Mikrobiologie und Hygiene, Freiburg, Germany.
Clinical specimens.
The PCR-EIA was evaluated with 16 clinical specimens. Lymph node biopsy specimens were obtained from nine
patients with clinically, serologically, and histopathologically proven
CSD. Two skin biopsy specimens were obtained from human
immunodeficiency virus-infected patients with histopathologically
diagnosed BA. Five lymph node specimens from patients without any
evidence of CSD were used as negative controls.
Extraction of DNA.
DNA was extracted from the (mostly
formalin-fixed, paraffin-embedded) lymph node and skin biopsy specimens
by using a commercially available kit (Qiagen GmbH, Hilden, Germany) as
proposed by the manufacturer. The extracted DNA was used as a template
in the PCR assays. Purified DNAs from cultured bacterial strains of
B. henselae and B. quintana were used as positive
controls. Extracted DNA from non-Bartonella species and from
five lymph node specimens from patients without evidence of CSD were
used as negative controls.
Primers and probes.
The primer pair previously described by
Relman et al. (17) was used to amplify a 296-bp fragment of
the Bartonella 16S rRNA gene by PCR as described elsewhere
(20). PCR products were labeled during the amplification by
using the 5'-biotin-modified primer p12B-bio (Table
1). Digoxigenin end-labeled B. henselae-specific (RHp-dig) and B. quintana-specific
(RQp-dig) oligonucleotide probes (Table 1) were prepared according to
the sequence given by Daly et al. (8) and were used for the
detection of PCR products by dot blot hybridization and EIA. The probes
differed from each other by three nucleotides.
PCR amplification.
The reaction mixture consisted of bovine
serum albumin (BSA; 8 ng/µl), deoxynucleoside triphosphates (200 µM
each), primers (117 nM each), Taq polymerase (4 U; Pharmacia
Biotech), and 5 µl of extracted DNA in 100.0 µl of TBE
(Tris-borate-EDTA) buffer. The PCR was performed as described
previously (20).
All oligonucleotides were synthesized and were modified at their 5'
ends with biotin or digoxigenin by Birsner & Grob Biotech
(Freiburg,
Germany).
EIA for detection of PCR products.
The EIA was performed as
described previously by Lüneberg et al. (14) with
modifications. Streptavidin-coated microtiter plates (Micro Coat GmbH,
Penzberg, Germany) were washed two times with phosphate-buffered saline
(PBS)-0.05% Tween. All aliquots of the PCR mixtures were analyzed in
duplicate. The PCR fragments were generated with the biotinylated
primer p12B-bio and the unlabeled primer p24E. A total of 12 µl of
the PCR product was diluted in 10 mM sodium phosphate (pH 7.4)-100 mM
NaCl to a final volume of 60 µl and was pipetted into each well for
immobilization on streptavidin-coated microtiter wells. After binding
of the PCR product via the incorporated biotinylated primer for 15 min,
the wells were washed three times with PBS-0.05% Tween. The
double-stranded PCR product was denatured with 0.1 M NaOH for 10 min.
The unlabeled strands were removed by washing once with 0.1 M NaOH and
three times with 0.1 M Tris-HCl (pH 7.5; Merck, Darmstadt, Germany). The immobilized single-stranded PCR product was hybridized with 0.2 pmol of 5'-digoxigenin-labeled oligonucleotide RHp-dig or RQp-dig for
2 h in a water bath at 55°C. The hybridization solution contained 0.6 M NaCl, 20 mM sodium phosphate (pH 7.4), 1 mM EDTA (Merck, Darmstadt, Germany), 0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 0.02% BSA (Sigma, Deisenhofen, Germany), and 0.2 pmol of the digoxigenin-labeled oligonucleotide per well. Afterward,
the plates were washed three times with 6× SSC (1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate; Sigma) at room temperature and twice for 5 min in 3× SSC at 55°C in a water bath. Anti-digoxigenin Fab
fragments conjugated with horseradish peroxidase (Boehringer Mannheim)
were diluted at a final concentration of 150 mU/ml (1:1,000 in 1%
BSA-PBS), and 50 µl of this dilution was applied to the wells for 30 min at 37°C. The unbound conjugate was removed by washing with
PBS-0.05% Tween three times. Finally, 50 µl of
2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid (ABTS);
Boehringer Mannheim) substrate solution was added, and the mixture was
incubated at 37°C with shaking for 30 min. The
A405 of each well was determined with a
microtitration plate reader (Ceres 900; Biotek).
Analysis of PCR products by dot blot hybridization.
Hybridization of the PCR products was performed by the protocol
described by Anderson et al. (1), with modifications. The internal digoxigenin-labeled oligonucleotide probes RHp-dig and RQp-dig
used as hybridization probes were the same as those used for PCR-EIA.
In short, 10 µl of each PCR product was denatured for 5 min at 95°C
and was then put immediately on ice. Five-microliter aliquots were
spotted onto each of two nylon membranes (Qiabrane Nylon plus; Qiagen
GmbH, Hilden, Germany) and air dried, and the DNA was cross-linked to
the nylon membrane by UV irradiation for 3 min. The membranes were then
blocked for 1 h at 61°C by using standard prehybridization
solution. Hybridization was performed for 1 h at 61°C. The
hybridization buffer contained 0.75 pmol/2 ml of probe RHp-dig for
detection of B. henselae or 7.5 pmol/2 ml of probe RQp-dig
for detection of B. quintana. The hybridized membranes were
washed twice for 15 min at 61°C in 5× SSC-0.1% sodium dodecyl
sulfate (SDS) to remove the surplus oligonucleotide probes. The
hybridized membrane was then washed, blocked, incubated for 30 min with
alkaline phosphatase-conjugated anti-digoxigenin-antibody (Boehringer
Mannheim), washed again twice for 15 min, and soaked in Luminogen CSPD
substrate (Boehringer Mannheim). To increase the luminescence reaction,
the membrane was stored for 25 min at 37°C. Afterward the membrane
was exposed to X-ray film (Kodak S-Omat AR) for 40 min, followed by
development of the film.
| |
RESULTS |
PCR amplification.
With the primers described by Relman et al.
(17), the DNAs of all Bartonella species known to
cause human diseases (B. henselae, B. quintana,
B. bacilliformis, B. elizabethae, B. clarridgeiae) could be amplified (Fig.
1). No differences in PCR amplification were observed by using the biotinylated or the unbiotinylated oligonucleotide primer p12B. None of the other
non-Bartonella bacterial strains reacted (Table
2).
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FIG. 1.
Agarose gel electrophoresis of the amplified DNAs of
five Bartonella species and Afipia felis, which
is known to cause human infections. Lane 1, molecular size marker; lane
2, negative control; lanes 3 and 4, B. quintana; lanes 5 and
6, B. henselae; lanes 7 and 8, B. elizabethae;
lanes 9 and 10, B. bacilliformis; lanes 11 and 12, B. clarridgeiae; lanes 13 and 14, A. felis.
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TABLE 2.
Specificity and sensitivity of PCR, PCR-EIA, and dot blot
hybridization on the basis of results with serial dilutions of B. henselae and B. quintanaa
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EIA for detection of PCR products.
All incubation steps and
concentrations of reagents were optimized with the strains of B. henselae and B. quintana mentioned above. An optical
density (OD) by the PCR-EIA of 
0.2 was a considered positive result
and corresponded to twice the absorbance for the negative control. With
this cutoff, B. henselae and B. quintana could be
detected with the oligonucleotides RHp-dig and RQp-dig down to a
concentration of 103 CFU/ml in serial dilutions,
corresponding to an equivalent of 5 CFU of Bartonella
species in the PCR mixture with 5 µl of DNA (Table 2). No
cross-reactivity was observed between B. henselae and
B. quintana or with the other Bartonella species.
Thus, the oligonucleotides RHp-dig and RQp-dig appear to be specific
for B. henselae and B. quintana,
respectively. None of the non-Bartonella isolates hybridized
with either probe.
Analysis of PCR products by dot blot hybridization.
The same
oligonucleotide probes used for detection of B. henselae
(RHp-dig) and B. quintana (RQp-dig) by PCR-EIA were also applied in the dot blot hybridization assay by using the same serial
dilutions of both Bartonella strains. By dot blot
hybridization both probes were specific for B. henselae and
B. quintana, and no cross-reactivity with the
Bartonella species or non-Bartonella isolates was
seen (Table 2).
Determination of sensitivities of PCR-EIA and dot blot
hybridization.
One aliquot of each of the 10-fold dilutions of
suspensions of B. henselae and B. quintana was
used for quantitative culture to determine the number of CFU, while
another aliquot was analyzed in parallel by the PCR-based methods. In
ethidium bromide-stained agarose gels, a band was visible from PCRs
performed with 1 to 5 µl of dilutions of up to 103 CFU of
B. henselae and B. quintana per ml; these
corresponded to 1 to 5 CFU in the reaction mixture. The sensitivity
obtained by the PCR-EIA was approximately 10-fold higher; weakly
positive bands in the agarose gels gave a clear positive result in the EIA. Dot blot hybridization was also 10 times more sensitive than the
test with ethidium bromide-stained gels (Table 2).
PCR-EIA and hybridization with clinical specimens.
From the 16 clinical specimens which were tested by both PCR-EIA and dot blot
hybridization, the lymph nodes from five control patients (without
Bartonella infections) did not react by either method. The
results for specimens from 11 patients with suspected Bartonella infection are shown in Fig.
2 and Table
3. The skin biopsy specimens from the two
patients with BA reacted only with the B. quintana-specific
probe by both methods, whereas the 9 lymph node biopsy specimens from
patients with CSD gave positive results with the B. henselae-specific probe. None of the five samples without
suspected Bartonella infection hybridized with either probe.
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FIG. 2.
Results of dot blot hybridization with the B. henselae-specific probe RHp-dig and the B. quintana-specific probe RQp-dig with PCR products of samples from
patients with BA (rows 1 and 2), patients with CSD (rows 3 to 11), and
negative control patients (rows 12 to 14) in three dilution steps: a,
1:1; b, 1:5; and c, 1:50. Dot Ka, negative control; dot Kb, a B. henselae strain; dot Kc, a B. quintana strain.
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The approximate numbers of CFU in the clinical samples were calculated
as follows: with bacterial suspensions (Table
2) an
OD of 0.2 corresponded roughly to 10
3 CFU/ml, but the increase in the
OD was not a linear function
of the increase in the number of CFU in
bacterial dilutions. Therefore,
an empiric standard curve was drawn by
using the data given in
Table
2 to determine the concentrations in the
clinical specimens.
By using the results for dilutions of 1:1, 1:5, and
1:50 of the
clinical samples, the numbers of CFU were detected on the
basis
of the highest positive OD (Table
3). It should be emphasized
that these results are obtained by comparing the ODs obtained
by PCR
with DNA extracted from human specimens with the ODs obtained
by PCR
with DNA extracted from cultured bacteria, and differences
in the real
and the calculated bacterial densities in human specimens
cannot be
excluded.
| |
DISCUSSION |
Various PCR procedures with different target sequences for
detection of Bartonella species have been described (1,
3, 5, 10, 16, 21, 23). However, there are great differences in
the sensitivities of these procedures. The three target genes most
often used for PCR detection of Bartonella DNA are the 16S rRNA gene used in the present study, the citrate synthase gene (gltA), and the 60-kDa heat shock protein gene
(htrA). Few data are available to compare these assays. In a
recent study, the 16S rRNA gene PCR was slightly more sensitive than
the htrA gene PCR, but false-negative results were obtained
by both assays (21). However, agarose gel analysis and
ethidium bromide staining do not appear to be sensitive enough for
visualization of small amounts of PCR products. Dot blot hybridization
or enzyme immunoassay used for the detection of the PCR-amplified DNA
showed a 10-fold higher sensitivity than ethidium bromide staining in
some studies (1, 9, 11, 14, 18). Fujita et al.
(9) demonstrated that the OD of the EIA correlated with the
number of C. albicans blastoconidia suspended in blood.
Jantos et al. (12) developed a PCR-EIA for detection of
Chlamydia pneumoniae which was as sensitive as Southern blot
hybridization for the detection of PCR products and 100 times more
sensitive than visualization of PCR products on agarose gels.
Additionally, DNA extracted from human specimens may contain large
amounts of cellular DNA that may inhibit amplification. Therefore,
template dilution and amplification of different dilution steps may
increase the sensitivity of PCR (1, 11; our results).
Detection of B. henselae- and B. quintana-amplified DNA by EIA has been reported previously by
Ritzler and Altwegg (18), but no serial dilutions of
Bartonella suspensions with detection of the number of CFU
or clinical specimens have been investigated. When serial dilutions of
the bacterial suspensions or different dilution steps of the template
from clinical specimens were analyzed in our study, dot blot
hybridization and EIA were 10 times more sensitive than agarose gel
electrophoresis with ethidium bromide staining. We were able to detect
1 to 5 CFU of Bartonella species in the PCR mixture with
bacterial dilutions and in the clinical specimens corresponding to
1 × 103 to 2 × 106 CFU/ml. Burnie
et al. (6) described a semiquantitative PCR-EIA for the
detection of circulating DNA in patients with disseminated candidiasis
and showed that the OD also correlated with the clinical status of the
patients, becoming negative if therapy was successful and progressively
more positive if the patient's condition deteriorated. However, it
cannot be excluded that the OD and the calculated number of CFU per
milliliter in our clinical specimens do not correspond exactly to the
number of CFU in the investigated bacterial dilutions. As already
mentioned, cellular DNA in human specimens may have an inhibitory
effect on the amplification of the bacterial DNA, and the real number
of CFU per milliliter in human specimens could be even higher.
In conclusion, our PCR-based EIA for the species-specific
identification and quantification of amplified B. henselae
and B. quintana DNAs was more sensitive than ethidium
bromide staining of agarose gels, less time-consuming, and less
labor-intensive than sequencing or dot blot hybridization for
identification of amplified DNA. Species-specific results can be
obtained within 24 h, and we think that this assay will improve
the means of diagnosis of Bartonella infections.
| |
ACKNOWLEDGMENTS |
We thank Wolfgang Bredt for helpful discussions and critical
reading of the manuscript and Karin Oberle for excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Medizinische Mikrobiologie und Hygiene,
Hermann-Herder-Str. 11, D-79104 Freiburg, Germany. Phone: (49)
761-203-6529. Fax: (49) 761-203-6562. E-mail:
sander{at}ukl.uni-freiburg.de.
| |
REFERENCES |
| 1.
|
Anderson, B.,
K. Sims,
R. Regnery,
L. Robinson,
M. J. Schmidt,
S. Goral,
C. Hager, and K. Edwards.
1994.
Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR.
J. Clin. Microbiol.
32:942-948[Abstract/Free Full Text].
|
| 2.
|
Anderson, B. E., and M. A. Neuman.
1997.
Bartonella spp. as emerging human pathogens.
Clin. Microbiol. Rev.
10:203-219[Abstract].
|
| 3.
|
Avidor, B.,
Y. Kletter,
S. Abulafia,
Y. Golan,
M. Ephros, and M. Giladi.
1997.
Molecular diagnosis of cat scratch disease: a two-step approach.
J. Clin. Microbiol.
35:1924-1930[Abstract].
|
| 4.
|
Bass, J.,
J. M. Vincent, and D. A. Person.
1997.
The expanding spectrum of Bartonella infections. II. Cat-scratch disease.
Pediatr. Infect. Dis. J.
16:163-179[Medline].
|
| 5.
|
Bergmans, A. M.,
J. W. Groothedde,
J. F. P. Schellekens,
J. D. A. van Embden,
J. M. Ossewaarde, and L. M. Schouls.
1995.
Etiology of cat scratch disease: comparison of polymerase chain reaction detection of Bartonella (formerly Rochalimaea) and Afipia felis DNA with serology and skin tests.
J. Infect. Dis.
171:916-923[Medline].
|
| 6.
|
Burnie, J. P.,
N. Golbang, and R. C. Matthews.
1997.
Semiquantitative polymerase chain reaction enzyme immunoassay for diagnosis of disseminated candidiasis.
Eur. J. Clin. Microbiol. Infect. Dis.
16:346-350[Medline].
|
| 7.
|
Cáceres-Ríos, H.,
J. Rodríguez-Tafur,
F. Bravo-Puccio,
C. Maguina-Vargas,
C. Sanguineti Díaz,
D. C. Ramos, and R. Patarca.
1995.
Verrugua peruanan: an infectious endemic angiomatosis.
Crit. Rev. Oncog.
6:47-56[Medline].
|
| 8.
|
Daly, J. S.,
M. G. Worthington,
D. J. Brenner,
C. W. Moss,
D. G. Hollis,
R. S. Weyant,
A. G. Steigerwalt,
R. E. Weaver,
M. I. Daneshvar, and S. P. O'Connor.
1993.
Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis.
J. Clin. Microbiol.
31:872-881[Abstract/Free Full Text].
|
| 9.
|
Fujita, S.,
B. A. Lasker,
T. J. Lott,
E. Reiss, and C. J. Morrison.
1995.
Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.
J. Clin. Microbiol.
33:962-967[Abstract].
|
| 10.
|
Goldenberger, D.,
T. Schmidheini, and M. Altwegg.
1997.
Detection of Bartonella henselae and Bartonella quintana by a simple and rapid procedure using broad-range PCR amplification and direct single-strand sequencing of part of the 16S rRNA gene.
Clin. Microbiol. Infect.
3:240-245.
[Medline] |
| 11.
|
Goldenberger, D.,
R. Zbinden,
I. Perschil, and M. Altwegg.
1996.
Nachweis von Bartonella (Rochalimaea) henselae/B. quintana mittels Polymerase Kettenreaktion (PCR).
Schweiz. Med. Wochenschr.
126:207-213[Medline].
|
| 12.
|
Jantos, C. A.,
R. Roggendorf,
F. N. Wuppermann, and J. H. Hegemann.
1998.
Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay.
J. Clin. Microbiol.
36:1890-1894[Abstract/Free Full Text].
|
| 13.
|
Kordick, D. L.,
E. J. Hilyard,
T. L. Hadfield,
K. H. Wilson,
A. G. Steigerwald,
D. J. Brenner, and E. B. Breitschwerdt.
1997.
Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease).
J. Clin. Microbiol.
35:1813-1818[Abstract].
|
| 14.
|
Lüneberg, E.,
J. S. Jensen, and M. Frosch.
1993.
Detection of Mycoplasma pneumoniae by polymerase chain reaction and nonradioactive hybridization in microtiter plates.
J. Clin. Microbiol.
31:1088-1094[Abstract/Free Full Text].
|
| 15.
|
Margileth, A. M., and D. F. Baehren.
1998.
Chest-wall abscess due to cat-scratch disease (CSD) in an adult with antibodies to Bartonella clarridgeiae: case report and review of the thoracopulmonary manifestation of CSD.
Clin. Infect. Dis.
27:353-357[Medline].
|
| 16.
|
Mouritsen, C. L.,
C. M. Litwin,
R. L. Maiese,
S. M. Segal, and G. H. Segal.
1997.
Rapid polymerase chain reaction-based detection of the causative agent of cat scratch disease (Bartonella henselae) in formalin-fixed, paraffin-embedded samples.
Hum. Pathol.
28:820-826[Medline].
|
| 17.
|
Relman, D. A.,
J. S. Loutit,
T. M. Schmidt,
S. Falkow, and L. S. Tompkins.
1990.
The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.
N. Engl. J. Med.
323:1573-1580[Abstract].
|
| 18.
|
Ritzler, M., and M. Altwegg.
1996.
Sensitivity and specificity of a commercially available enzyme-linked immunoassay for the detection of polymerase chain reaction amplified DNA.
J. Microbiol. Methods
27:233-238.
|
| 19.
|
Sander, A.
1998.
Microbiological diagnosis of Bartonella species and Afipia felis, p. 98-112.
In
A. Schmidt (ed.), Contributions to microbiology, vol. 1. Bartonella and Afipia species emphasizing Bartonella henselae. Karger, Basel, Switzerland.
|
| 20.
|
Sander, A.,
C. Bühler,
K. Pelz,
E. von Cramm, and W. Bredt.
1997.
Detection and isolation of two Bartonella henselae variants in domestic cats in Germany.
J. Clin. Microbiol.
35:584-587[Abstract].
|
| 21.
|
Sander, A.,
M. Posselt,
N. Böhm,
M. Ruess, and M. Altwegg.
1999.
Detection of Bartonella henselae DNA by two different PCR assays and determination of the genotype in histologically defined cat scratch disease.
J. Clin. Microbiol.
37:993-997[Abstract/Free Full Text].
|
| 22.
|
Sander, A.,
M. Posselt,
K. Oberle, and W. Bredt.
1998.
Seroprevalence of antibodies to Bartonella henselae in patients with cat scratch disease and in healthy controls: evaluation and comparison of two commercial serological tests.
Clin. Diagn. Lab. Immunol.
5:486-490[Abstract/Free Full Text].
|
| 23.
|
Scott, M. A.,
T. L. McCurley,
C. L. Vnencak-Jones,
C. Hager,
J. A. McCoy,
B. Anderson,
R. D. Collins, and K. M. Edwards.
1996.
Cat scratch disease. Detection of Bartonella henselae DNA in archival biopsies from patients with clinically, serologically, and histologically defined disease.
Am. J. Pathol.
149:2161-2167[Abstract].
|
Journal of Clinical Microbiology, October 1999, p. 3097-3101, Vol. 37, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
