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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.8.3800–3806.2005

Diagnosis of Cat Scratch Disease with Detection of

Bartonella henselae

by PCR: a Study of Patients with Lymph Node Enlargement

Yves Hansmann,

* Sylvie DeMartino,

Yves Pie

´mont,

Nicolas Meyer,

Philippe Mariet,

Re

´my Heller,

Daniel Christmann,

and Benoıˆt Jaulhac

Service des Maladies Infectieuses et Tropicales,1Laboratoire de Bacte´riologie,2and Departement de Sante´ Publique,3

Hoˆpitaux Universitaires de Strasbourg, Strasbourg, France

Received 22 October 2004/Returned for modification 7 January 2005/Accepted 16 March 2005

Cat scratch disease (CSD) is mostly due toBartonella henselaeafter inoculation of the organism through a
skin injury. Since the causative bacteria cannot be easily cultured from human lymph node samples, the
diagnosis usually relies on epidemiological, clinical, histological, and serological criteria (classical criteria). A
study was performed to determine the diagnostic value of PCR analysis for the detection ofB. henselaefor the
diagnosis of CSD and its place in the diagnostic strategy alongside the classical criteria. Over a 7-year period,
lymph node biopsy specimens or cytopunctures from 70 patients were systematically tested by PCR for the
presence of B. henselaeDNA (htrAgene) in the Bacteriology Laboratory of the Hoˆpitaux Universitaires de
Strasbourg. Serological testing by an immunofluorescence assay forB. henselaeantibodies was also performed
for each patient, and clinical, epidemiological, and histological data were collected. The patients were then
divided into two groups according to the number of positive diagnostic criteria for CSD: 29 patients with
definite CSD (two or more classical criteria) and 15 patients with possible CSD (less than two classical
criteria). The remaining 26 patients for whom another diagnosis was retained were used as a control group.
Among all criteria, PCR analysis had the best specificity (100%). The PCR assay forB. henselaewas positive
for 22 (76%; 95% confidence interval [CI95], 56.5 to 89.7%) of the 29 definite CSD patients and 3 (20%; CI95,

4.3 to 48.1%) of the 15 possible CSD patients. We then studied combinations of diagnostic criteria, including

B. henselaePCR analysis. The best diagnostic performance was observed if at least two criteria were present
among serologic, epidemiologic, histological, and molecular criteria.

Cat scratch disease (CSD) is the most frequent clinical
man-ifestation of Bartonella infections in immunocompetent
pa-tients (8, 9, 18, 19, 23, 28).Bartonella henselae, the main
caus-ative agent of CSD, can be detected in the blood of healthy cats
(15), and cats can transmitBartonellato humans after a skin
injury caused by a scratch or bite (19). The disease was first
described by Debre´ et al. in 1950 on the basis of
epidemiolog-ical and clinepidemiolog-ical data (8), and the causality ofB. henselae in
CSD has since been demonstrated by serological and
molecu-lar assays (4, 23, 24, 25, 29).

CSD appears as regional lymph node enlargement after a cat
scratch or bite in the same area. The clinical manifestations
include inflammatory lymphadenopathy, which appears 1 to 7
weeks after the injury, and a papular lesion of the skin, which
develops at the site of the injury. The diagnostic challenge for
the physician is to prove or invalidate the CSD etiology in the
face of a patient with lymph node enlargement. In most cases,
the diagnosis is based on a combination of clinical,
epidemio-logical, seroepidemio-logical, and histological data. According to
Berg-mans et al. (5), a diagnosis of CSD usually requires three of the
following four criteria: (i) a history of contact with a cat and
the presence of a scratch or primary lesion of the skin, eye, or
mucous membrane; (ii) a positive cat scratch skin test reaction;
(iii) negative laboratory testing for other causes of

lymphade-nopathy; and (iv) characteristic histopathological findings in a
lymph node biopsy specimen or at a site of systemic
involve-ment. However, none of these criteria are sufficiently specific
to establish a diagnosis of CSD. In addition, the CSD
intra-dermal skin test (8) is no longer available, and a history of a cat
injury is sometimes not reported by the patient. Another
pos-sibility for diagnosis is histological examination of the lymph
node involved; but this cannot differentiate between several
infectious etiologies, including tularemia, bartonellosis, and
other inoculation diseases. A typical CSD histology showing a
granuloma with central necrosis, multinucleated giant cells,
and microabscesses may also be absent. Thus, histological
ex-amination at an early stage of the disease in fact shows only
lymphoid hyperplasia and arteriolar proliferation. Conversely,
in the presence of granuloma, a differential diagnosis with
respect to tuberculosis or other infectious diseases that display
granulomas can be very difficult. Serology fails in terms of
specificity and/or sensitivity (5, 10), while culture ofB. henselae

from lymph node tissue samples is difficult and has been
re-ported in only a very limited number of cases (18). The
detec-tion of specific DNA fragments by PCR has been proposed as
a novel method for demonstration of the presence of B.
henselaein CSD (3, 4, 5, 18, 21, 22, 25). Its utility nevertheless
remains to be assessed, and there is still no established “gold
standard” for the diagnosis of cat scratch disease.

The aim of our study was to determine the diagnostic value
ofB. henselaedetection by PCR in CSD and the place of PCR

among the other usual diagnostic tools. An observational study
was conducted on the basis of data collected prospectively

* Corresponding author. Mailing address: Service des Maladies
In-fectieuses et Tropicales, Hoˆpitaux Universitaires de Strasbourg, 1,
Place de l’Hoˆpital, 67091 Strasbourg Cedex, France. Phone: 33 3 88 11
53 51. Fax: 33 3 88 11 64 64. E-mail: Yves.Hansmann@chru-strasbourg
.fr.

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from patients with inflammatory lymph node enlargement
re-quiring biopsy or adenectomy.

MATERIALS AND METHODS

Patients.Over the period from 1993 to 2000, we prospectively collected the
following general and clinical data from every patient consulting at the Hoˆpitaux
Universitaires de Strasbourg for local superficial lymphadenopathy and for
whom a cytopuncture or biopsy of the lymph node was performed: age, gender,
medical history, localization of the lymph nodes affected, contact with domestic
or wild animals, and the presence of a scratch or bite by a domestic or wild
animal and its site. Part of the lymphoid tissue was sent to the Bacteriology
Laboratory for testing for the usual bacteria and PCR assay forB. henselae. In
the case of biopsy samples, another portion was subjected to a histological
examination. For each patient, a standard serodiagnostic test forB. henselaewas
performed in the Bacteriology Laboratory of the Hoˆpitaux Universitaires de
Strasbourg. All patients were reexamined after 2 to 6 months to record the
evolution of the adenopathy and determine the validity of the CSD diagnosis.
Finally, we focused our study on 44 patients who had provided complete data
concerning their medical history and contact with animals and for whom serology
testing and PCR analysisB. henselaehad been carried out in our laboratory.

For these 44 patients with lymphadenopathy, the final diagnosis of CSD was
made or not on the basis of the presence or the absence of the following three
additional criteria: (i) close contact with cats or a scratch or bite from a cat, (ii)
a typical CSD histology, i.e., granuloma with a central pyogenic abscess
(lym-phoid hyperplasia not being sufficiently specific to establish a diagnosis of CSD),
and (iii) positive serology by an immunofluorescence assay for antibodies against

B. henselae. These 44 patients were thus divided into two groups, as follows: the
first group of 29 patients was classified as definitely having CSD, according to the
presence of at least two of the above three criteria, and the second group of 15
patients had possible CSD and presented with only one or none of the criteria for
CSD given above.

Twenty-six lymph node samples from patients for whom a diagnosis other than
CSD had been established on the basis of histological criteria or bacteriological
tests (positive serology or bacterial or mycobacterial cultures) were used as
negative controls.

DNA extraction.The lymph node specimens were cut into small pieces by
using a sterile scalpel blade. Approximately 40 mg of tissue was then washed
twice in 0.5 ml of sterile phosphate-buffered saline (PBS); and the tissue was
treated with 500␮g/ml of proteinase K (Sigma) in 1 ml of 10 mM Tris HCl (pH
8.0) containing 0.5% Nonidet P-40 (Sigma), 0.5% Tween 20, 50 mM KCl, and 50
mM MgCl2at 55°C and with 30 s of vigorous shaking every 15 min for 2 h or until
the tissue was entirely digested. The DNA was extracted with phenol-chloroform,
ethanol precipitated, air dried, resuspended in 40␮l of TE buffer (10 mM Tris
HCl, pH 8.0, 1 mM EDTA), and heated to 95°C for 10 min. A 3-␮l aliquot of this
suspension was amplified by PCR.

PCR primers and hybridization probe.The detection ofB. henselaein CSD
lymph nodes with the primers and internal hybridization probe employed in this
study has previously been described by Anderson et al. (3). These primers target
a 414-bp fragment in thehtrAgene ofB. henselae.

DNA amplification.A 3-␮l aliquot of the DNA suspension extracted from a
lymph node tissue sample was used as the template for 40 cycles of DNA
amplification. PCR amplification was performed in 20␮M Tris HCl (pH 8.4)
containing 50 mM KCl, 3 mM MgCl2, 1.5 units ofTaqpolymerase (Invitrogen,
Cergy Pontoise, France), 0.2␮M of each primer, and 0.2␮M of each of the four
deoxyribonucleotides in a final volume of 100␮l.

To avoid DNA contamination of the samples, the precautions recommended
by Kwok and Higuchi (17) were taken. Sample preparation, PCR amplification,
and electrophoresis were performed with separate sets of pipettes and the
wear-ing of protective laboratory coats and caps in three different closed rooms where

B. henselaehad never been cultured. At each step of sample preparation, each
tube was carefully and separately uncovered. Gloves were changed between the
handling of each sample, and all solutions were manipulated by using pipettes
with hydrophobic filter tips (Multiguard; Sorenson).

In each run of four coded tissue samples, three negative controls were added.
The first consisted of the reaction mixture without any DNA template, while the
second contained DNA from a strain ofBartonellaother thanB. henselae. In
order to detect sample-to-sample contamination during DNA preparation, a
third control consisting of a 0.5-ml aliquot of a tissue sample from a patient with
a noninfectious disease was blindly and simultaneously processed and amplified
with the four other samples.

To monitor the DNA amplification efficiency, a positive control containing 1
pg of purifiedB. henselaeDNA (ATCC 49882) was included in each run. All

positive samples were checked by processing and amplification of another frozen
aliquot of the same tissue specimen. All negative samples were amplified again
after addition of 1 pg of purifiedB. henselaeDNA, in order to detect a possible
inhibitor of the amplification reaction.

PCR amplification was performed in an Applied Biosystems 9700 thermal
cycler. After predenaturation for 3 min at 94°C, samples were amplified through
40 cycles of 93°C for 30 s, 55°C for 30 s, and 72°C for 60 s, followed by a final
extension step of 8 min at 72°C.

A 10-␮l aliquot from each PCR tube was electrophoresed through a 3%
agarose NuSieve containing 1% SeaKem agarose gel (FMC Bioproducts) for
1.5 h at 120 V. DNA was transferred onto a positively charged nylon membrane
(Roche, Meylan, France) and fixed for Southern blotting according to the
man-ufacturer’s recommendations. The membranes were prehybridized for 30 min at
55°C in 6⫻SSPE buffer (1⫻SSPE buffer is 0.18 M NaCl, 10 mM NaH2PO4, and
1 mM EDTA [pH 7.7]) supplemented with 0.02% bovine serum albumin, 0.02%
Ficoll 400, and 0.02% polyvinylpyrrolidone and then transferred into fresh
hy-bridization buffer containing 0.5 pmol/ml of the internal probe 5⬘labeled with
[␥-32

P]ATP. Hybridization was performed for 2 h at 45°C and was followed by
two washes at 30°C for 10 min in 2⫻SSPE buffer containing 0.1% sodium
dodecyl sulfate. After the membranes were air dried, they were exposed
over-night at–70°C to an X-ray film (Fuji) with two intensifying screens.

Serology (2).B. henselae(ATCC 49882) grown on Vero cells (ATCC CCL-81)

was used to prepare the antigen. The ATCC 49882 strain was previously
com-pared to three otherB. henselaestrains isolated from stray cats (13) as the
antigen source for indirect immunofluorescence assays and was shown to be
superior or equivalent to the other strains tested for both specificity and
sensi-tivity (data not shown). The cells were first cultured in 25-cm2flasks 
(Corning-Costar, Brumath, France) as a confluent unicellular layer in the presence of 89%
Dulbecco’s modified Eagle’s medium (Invitrogen, Cergy Pontoise, France), 1%
0.2 M glutamine solution (Merck, Nogent sur Marne, France), and 10% fetal calf
serum (Seromed, Berlin, Germany). The cultures were incubated at 35°C under
5% CO2and the confluent cellular layer was dissociated with 0.05% trypsin in
0.53 M EDTA solution.

The cells were then infected by addition of 5 ml of a 0.5 to 1 McFarland
suspension ofB. henselaepreviously grown on Columbia agar (Becton Dickinson,
Meylan, France) enriched with 5% rabbit blood for 6 days at 35°C under 5%
CO2. After 3 days, the infected cells were washed in prewarmed sterile PBS.
Uninfected Vero cells were cultured in parallel for use as controls. Infected and
uninfected cells were dissociated with 0.05% trypsin in 0.53 M EDTA. The cells
were resuspended in fresh culture medium and centrifuged at 200⫻gfor 10 min.
This washing step was repeated once, after which the pellets were resuspended
in fresh culture medium and the cell concentration was adjusted to 500 to 700⫻
103cells/ml. These cell suspensions were layered onto immunofluorescence assay
slide wells and incubated at 35°C under 5% CO2for 16 to 18 h. The cells were
then fixed on the slides with cold acetone for 15 min.

In the indirect immunofluorescence assays, the human sera to be tested for the
presence ofB. henselaeantibodies were diluted in PBS and incubated on the
fixed bacterial antigens for 30 min at 37°C. After the slides were washed for 10
min in PBS containing 1% (wt/vol) bovine serum albumin (Sigma), the slides
were incubated with fluorescein isothiocyanate-conjugated goat anti-total human

immunoglobulins (Fluoline H; BioMe´rieux, Marcy l’Etoile, France) previously
diluted to 1/50 in PBS. The slides were mounted in buffered glycerol (Fluoprep;
BioMe´rieux) and read under an Olympus fluorescence microscope at⫻400
magnification. The titer of a serum sample was defined as the highest dilution
that still showed for 50% of the infected cells a fluorescence intensity equal to the
highest intensity displayed by the positive control serum. Titers of⬍1/32 were
considered negative, titers ofⱖ1/64 were considered positive (sensitivity, 0.70;
specificity, 0.95), and titers of 1/32 were considered uncertain.

Statistical analysis.For a proportion, the 95% exact confidence interval (CI95)
was computed by using the binomial distribution. The comparison of localization
of lymphadenopathy for each diagnostic group was done by the
Fisher-Freeman-Halton test, which extends the Fisher exact test for tables with more than two
rows and/or columns. The alpha level was set at 5%, and the test was bilateral.

RESULTS

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The lymph node enlargements were mainly axillar in the
definite CSD group (51.7% of patients; CI95, 32.5 to 70.6%)

and, less frequently, inguinal or cervical (24.1% of patients in
both cases; CI95, 10.3 to 43.5%) (Table 1). However, inguinal

lymph node enlargement was observed more often in
individ-uals in the definite CSD group than in those in the other two
groups. In the possible CSD group, axillar and cervical lymph
node enlargements were found at approximately the same
fre-quency (40 to 50%). Cervical lymph node enlargement was
most commonly observed in the negative control group
(61.5%; CI95, 40.6 to 79.8%) (Table 1).

In the definite CSD group, analysis of the number of
classi-cal criteria for CSD (Table 2) revealed that 26 of the 29
patients (89.7%; CI95, 72.6 to 97.8%) had experienced a cat

scratch or had been in contact with cats, one had been injured
by a monkey, and the two remaining patients did not recall any
animal contact. Among the 19 patients in whom a histological
examination of the lymph node was performed, 16 (84.2%;
CI95, 60.4 to 96.6%) presented with a pyogenic granuloma and

the three presented with other nonspecific lymphocytic
inflam-mation. Serological testing forB. henselaeantibodies was
pos-itive for 25 of these 29 individuals (86.21%; CI95, 68.3 to

96.1%). PCR assay forBartonellawas positive for 22 patients
(sensitivity, 0.76; CI95, 56.5 to 89.7%) (Table 2), while all 7

patients from this group with a negative PCR result (6 of them
had a histological examination) were positive for only two of
the three classical criteria for CSD.

The possible CSD group, which comprised 15 patients who
presented with only one or no criteria for CSD (Table 2), was

heterogeneous, with 4 individuals (26.7%; CI95, 7.8 to 55.1%)

having a history of cat contact. A histological examination was
performed for nine of these patients, and the result was never
compatible with CSD.B. henselaeserology was positive for five

patients (33.33%; CI95, 11.8 to 61.6%), while only three

pa-tients (20%; CI95, 4.3 to 48.1%) had a positive PCR assay

result. The latter set of patients always displayed at least one
classical criterion for CSD: two patients had a history of
con-tact with cats, and one was positive for CSD serology. A
his-tological analysis of the lymph node was not available for any
of these three individuals because these three samples were
pus samples. Six patients in whom no other final diagnosis had
been retained presented no CSD criteria. All patients in this
group had good evolution of their lymph node enlargement 2
to 6 month after diagnosis.

For the 26 negative controls for whom another diagnosis had
been established, the causes of adenopathy are shown in Table
3. These individuals mainly presented with noninfectious
adeno-pathy (17 patients), including 8 cases of lymphoma, 4 cases of
benign tumor, and 2 cases of carcinoma. Among the nine
patients with infectious adenopathy, three had tuberculosis, as
revealed byMycobacterium tuberculosis-positive tissue cultures
and/or histological necrosis with caseum; three had pyogenic
adenitis due to Staphylococcus aureuswith histologically
evi-dent, acute purulent inflammation of the lymph node; and
three had serologically confirmed tularemia.

Analysis of the distribution of the classical criteria (Table 2)
for the 26 patients in the negative control group revealed that
9 mentioned contact with cats but only 1 had experienced a cat
scratch before the appearance of adenopathy. TheB. henselae

serology was tested in 21 patients in this group, and 3 of them
were positive with a titer ofⱖ1/64, but the final diagnosis for

TABLE 1. Clinical data for the patients in each diagnostic group
Characteristic Definite CSD

group

Possible
CSD group

Control
group

Mean age (yr [interval]) 26.72 (1–64) 19.6 (1–62) 30.84 (1–67)
Sex ratio (no. of F/no. of M)a 12/17 6/9 12/14
Localization of adenopathy

(no. of patients)

Axillar 15 6 6

Cervical 7 7 16

Inguinal 7 2 3

Multiple sites 0 0 1

Totalb 29 15 26

a

F, female; M, male.

b

Fischer-Freeman-Halton test,P⫽0.0650.

TABLE 2. Number of positive CSD criteria for the patients in each diagnostic group

Diagnosis Total no.

of patients

No. of patients positive for the following criteria/total no. of patients tested (%):

History of
contact with cats

Presence of

B. henselaeantibodies

PositiveB. henselae

PCR assay result

Histology compatible
with CSD

Definite CSDa 29 26/29 25/29 22/29 (75.9)c 16/19

Possible CSDb 15 4/15 5/15 3/15 (20)d 0/9

Other causes of adenopathy 26 1/26 3/21 0/26e 3/23

aPresence of at least two criteria among a history of cat contact, presence ofB. henselaeantibodies, and histology compatible with CSD.
bPresence of less than two of the criteria described in footnotea.

cCI

95, 56.5 to 89.7%.
dCI

95, 4.3 to 48.1%.
eCI

95, 0 to 13.2%.

TABLE 3. Etiologies of adenopathy for patients
in the control group

Diagnosis No. of

patients

Lymphoma ... 8

Benign tumor... 4

Tuberculosis... 3

Staphylococcus aureusadenitis ... 3

Tularemia ... 3

Adenocarcinoma ... 2

Sarcoma... 1

Sarcoidosis ... 1

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these patients was staphylococcal lymphadenitis. PCR assay for

B. henselae was negative for all 26 individuals in this group
(0%; CI95, 0 to 13.2%). A histological examination was

per-formed for 23 patients, and 7 cases showed signs of granuloma,
3 of which were histologically compatible with CSD. However,
the clinical data together with a positive specific serology led to
a diagnosis of tularemia in all three cases.

Culture ofB. henselaeon chocolate agar (Becton Dickinson)
enriched with IsoVitaleX was done in our study and was always
negative.

The sensitivity and specificity of the PCR assay were
calcu-lated by comparing the results ofBartonellaDNA testing for
patients in the definite CSD and control groups. The sensitivity

was 76% (CI95, 56.5 to 89.7%), and the specificity was 100%

(CI95, 86.7 to 100%). Moreover, good sensitivity was

main-tained whatever the type of sample analyzed. Thus, among the
five cases in the definite CSD group for whom only pus and not
tissue samples were tested forB. henselaeDNA, the PCR assay
was positive for four of them. The positive predictive value is
100% (CI95, 84.6 to 100%) if the control group is the patients

with a diagnosis of CSD by another means. The predictive
positive value is 88% (CI95, 68.8 to 97.5%) if the analysis is

done with the group with a possible diagnosis of CSD as the
control group.

In our study, 38 patients in the definite and possible CSD
groups had at least one of the previously defined CSD criteria
(Table 4). Among these, 29 displayed at least two of these
classical criteria and could be diagnosed as having definite
CSD. Among the 15 other possible CSD patients, the criteria
were insufficient to establish a diagnosis of CSD. On the other
hand, the good specificity and sensitivity observed for PCR
diagnosis of CSD allowed us to evaluate another combination
of criteria (enhanced diagnostic criteria) that included the
PCR assay as an additional factor (Table 4). By consideration
of all individuals positive for two of the enhanced diagnostic
criteria, we defined a group of 32 patients that comprised the
previous 29 in the initial definite CSD group and 3 others. The
PCR result was always associated with at least one other

cri-terion.

In this new group of 32 CSD patients, B. henselae PCR
testing was positive for 25 (78.1%; CI95, 60.0 to 90.7%) cases,

and hence, the sensitivity was 78%. By application of the same
enhanced criteria to the group of 12 patients for whom a
diagnosis of CSD had been excluded, 6 of these individuals had
one positive criterion for CSD, but this was never the PCR
result (CI95, 0 to 26.5%) (Table 4).

DISCUSSION

In this work, we used an efficient and specific PCR method
to detect theB. henselae htrAgene in lymph node tissues. We
chose to study patients selected on the basis of clinical
symp-toms compatible with a diagnosis of CSD. This allowed us to
determine the diagnostic value of the PCR assay in different
groups of patients classified according to the number of criteria
for CSD so as to be able to predict the sensitivity of the PCR
method with patients presenting with more or fewer
(some-times no) CSD criteria. Such an approach has, to our
knowl-edge, never been used before and is close to that used by a
physician who must make a diagnosis for a patient with
lymph-adenopathy with no etiological indication. Compared to the
classical diagnostic criteria for CSD, PCR analysis displayed
excellent specificity, since no false-positive results were
ob-served in our control group. The classical criteria nevertheless
remain useful, because the PCR result can sometimes be
neg-ative for patients with authentic CSD (7 of 29 patients in the

definite CSD group in our study).

The diagnosis of this disease relies on several criteria similar
to those originally described by Debre´ et al. (8). However, the
intradermal skin test is no longer available in several countries,
and in addition, none of the criteria initially used by Debre´ et
al. are etiologic markers of the disease (8). Thus, among the
classical criteria, neither a history of contact with cats nor a
clinical or histological examination alone is sufficient for the
diagnosis of CSD. A small minority of patients with cat
scratches develop CSD, and many cases of possible
CSD-re-lated adenopathy can be attributed to other causes. Similarly,
a histology picture compatible with CSD may be seen in other
conditions, such as tularemia, Nicolas Favre disease, or even
mycobacteriosis. New criteria which include serology and PCR
diagnosis should be of value for the diagnosis of an infection
due toB. henselae.

Serological testing for B. henselae antibodies was the first
microbiological test available but currently has a variable
pos-itive predictive value. It is an indirect diagnostic method which
can be negative in the early stage of the disease. In some
studies (7, 11, 28), the positive predictive value of the indirect
immunofluorescence assay forB. henselaewas reported to be
high (ⱖ91.4%). Conversely, Bergmans et al. (6) and Dupon et
al. (10) found a lack of sensitivity of the serological test among
patients with CSD.

On the other hand, both CSD serology and PCR assays
specific forB. henselaehave been reported to be negative (1, 3,

4, 5, 6, 12, 19, 28) in cases of authentic CSD, and the sensitivity
of PCR detection is often less than 80%. Among studies that
have tested well-defined cases of CSD, none have shown that
one PCR assay of a lymph node sample is sufficient for the
diagnosis of CSD. Avidor et al. (4) reported a sensitivity of

TABLE 4. Diagnosis of CSD by using the enhanced criteria
including the PCR result

Criterion and no. of criteria for
diagnosis of CSD

No. patients by use of:

Classical

criteriaa Enhanced

criteriab

Diagnosis of CSD retaineda

Three criteria 10 22

Two criteria 19 10

Total no. of patients with CSD 29 32
Diagnosis of CSD excludedb

One criterion 9 6

No criteria 6 6

Total no. of patients without CSD 15 12

a

Classical criteria for CSD diagnosis: (i) close contact with cats or a scratch or
bite from a cat, (ii) typical CSD histology (granuloma with a central pyogenic
abscess, with lymphoid hyperplasia not being sufficiently specific to establish a
diagnosis of CSD), and (iii) positive serology by an immunofluorescence assay
for antibodies againstB. henselae.

b

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100%, using three different PCR assays with three different
targets, but this is not current practice in routine diagnosis.

Several groups have already assessed the diagnostic value of
PCR analysis for CSD (1, 3, 4, 5, 6, 12, 20, 27). A comparison
of these studies is, however, difficult due to differences in the
PCR target, the sample type, and the criteria used to define
CSD. Thus, several primer pairs have been used to detectB.
henselaeby PCR amplification (3, 4, 5, 6, 14). The 16S rRNA
target first employed by Bergmans et al. (5) gave sensitivities of
96% among patients with a positive skin test result for CSD
and 60% among patients with a negative skin test result. In a
second study, the same authors (6) found that the sensitivities
were 86.4 and 100% for patients with more than two or more
than three criteria for CSD, respectively. ThehtrAgene used in

our study has frequently been employed to test clinical samples
among patients with suspected CSD. Anderson et al. (3) and
Goldenberger et al. (12) obtained sensitivities of 84 and 61%,
respectively, so that our result (sensitivity of 76%) is close to
the best for this target (3, 4). A comparison of the 16S rRNA
andhtrAtargets showed a better sensitivity of the former (60
versus 43%) (26). Avidor et al. (4) compared thegltA gene
(which encodes citrate synthase) with the 16S rRNA andhtrA

genes and found the first two targets to be more sensitive (100
and 94%, respectively) than thehtrAsequence (69%). Other
PCR targets were not tested in our work. However, the
spec-ificity of the results was ensured by processing and amplifying
a second aliquot for all the positive samples.

False-negative results can be explained either by a lack of
sensitivity, as suggested by the comparative studies of Avidor et
al. (4) and Sander et al. (25), or by the presence of other
species ofBartonella in CSD (13, 16, 21). A poor quality of
clinical samples without lymph node tissue or samples taken
after a long period of antibiotic therapy could also explain
some of these false-negative results. In most of the studies, the
samples were fresh lymph node biopsy specimens or pus drawn
from the lymph nodes (3, 4, 5, 6). Two other groups used fixed
paraffin-embedded lymph nodes (26, 27) and obtained
sensi-tivities of 40 to 70%, according to the amplification target and
the criteria used to define CSD.

A diagnosis of CSD must rely on the presence of a
combi-nation of epidemiological, histological, and bacteriological

cri-teria, since no single criterion may be considered the gold
standard. The criteria used to define CSD are hence of great
importance for estimation of the sensitivities and the
specific-ities of the biological tests used for its diagnosis, as has been
pointed out by several authors (6, 26, 27). Anderson et al. (3)
and Avidor et al. (4) selected patients with lymphadenopathy
with only contact with cats as the criterion for CSD. In our
study, the latter criteria misclassified one of our patients as
having CSD, although the patient in fact had pyogenic
ade-nopathy. The sensitivity of the PCR assay of Sander and Penno
(26) was 65% by the use of only histological criteria for case
definition and increased to 87% when serological results were
also considered, illustrating the low specificity of histological
criteria. In the study of Scott et al. (27), the patients were
selected because they fulfilled histopathological conditions and
were then analyzed according to different criteria. The
sensi-tivity of the PCR assay in that work was 68% (27). In our study,
histological evidence was present in 84% of the patients
dis-playing classical criteria but in only 72% of patients when the

enhanced criteria, including the PCR results, were used. There
were histological manifestations compatible with CSD in three
patients for whom this diagnosis was finally not retained. In our
study, we employed precisely defined clinical, serological,
ep-idemiological, and histological criteria. Our patients were
se-lected not only among those with a previously established
di-agnosis of CSD but also among all patients presenting with
lymphadenopathy and were divided into different groups,
ac-cording to the classical diagnostic criteria. This allowed us to
obtain a good estimation of the sensitivity of the PCR assay.

Goldenberger et al. (12) classified their patients into four
categories (certain CSD, possible CSD, unknown diagnosis,
and a control group) and tested miscellaneous samples, not all
of which were derived from cases of lymphadenopathy, and
obtained a sensitivity of 61% and a specificity of 100%. To
estimate the diagnostic value of our assay, especially for
pa-tients with uncertain CSD, we preferred to focus blindly on
cases of lymphadenopathy and to collect the data
prospec-tively, so as to define the different groups using the usual
criteria for CSD. We therefore determined the diagnostic
value ofhtrA PCR detection ofB. henselae as an additional
criterion for CSD and that of the expanded criteria that
in-cluded the PCR result. On the basis of our findings, only a
positive PCR assay result may be considered to be sufficiently
specific for the diagnosis of CSD, since no patient in the
con-trol group had a positive PCR test result, in contrast to the
results of serology (three false-positive results) and histology
(two false-positive results).

Adopting a clinical approach, we first determined the
diag-nostic value of PCR analysis for a group of patients fulfilling
the classical criteria for CSD. For such patients, the diagnosis
is generally easy to make. More interesting are patients who do
not fulfill all the criteria for CSD, for whom the diagnosis can
be very difficult and PCR assay ofB. henselaeis very helpful.
This situation is frequent in clinical practice: absent or
non-specific histolopathology, negative serology, or contact with
cats without any scratch, giving several combinations of
crite-ria. However, in our possible CSD patients who presented with

only one or none of the classical criteria but for whom no other
diagnosis could be retained, the B. henselae PCR assay was
positive in three cases. Insofar as these three patients always
displayed one of the classical criteria for CSD, we tested the
diagnostic value of the enhanced criteria (at least two criteria,
including the PCR result). By using these new criteria, a
diag-nosis of CSD was established for an additional 10% of patients.
Thus, by using the PCR assay as an additional criterion, the
sensitivity of CSD diagnosis could be improved without any
decrease in specificity, especially for patients with incomplete
diagnostic criteria. In our study, PCR detection ofB. henselae

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Other direct methods for detection ofBartonellainfections,
like immunohistochemical staining or culture, have been
re-ported for CSD diagnosis. These methods have not been used
in our work because of their lack of sensitivity and specificity.
Culture on chocolate agar enriched with IsoVitaleX was done
in our study and was always negative.

To establish a diagnosis of CSD in patients presenting with
superficial lymphadenopathy in one isolated area, we propose the
use of an etiological approach which consists of looking first for
the presence ofB. henselaeDNA by PCR analysis. In the case of
PCR positivity, CSD may be retained on account of the excellent
specificity. In the case of a negative PCR result, the diagnosis
could rely on the presence of at least two of the following criteria:
(i) positive serology, (ii) histology compatible with CSD (pyogenic
granuloma), or (iii) contact with cats during the days or weeks
preceding lymphadenopathy, together with elimination of any
other cause of lymph node enlargement (Fig. 1).

ACKNOWLEDGMENTS

We thank C. Barthel and E. Collin for their excellent technical
assistance.

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