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Journal of Clinical Microbiology, October 2001, p. 3743-3746, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3743-3746.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Diagnostic Yield of a PCR Assay in Focal
Complications of Brucellosis
P.
Morata,1
M. I.
Queipo-Ortuño,1
J. M.
Reguera,2
F.
Miralles,2
J. J.
Lopez-Gonzalez,3 and
J.
D.
Colmenero2,*
Department of Biochemistry and Molecular
Biology, Faculty of Medicine, Málaga
University,1 and Infectious Diseases
Unit, Internal Medicine Department,2 and
Microbiology Service,3 "Carlos Haya"
University Hospital, Málaga, Spain
Received 20 February 2001/Returned for modification 1 June
2001/Accepted 23 July 2001
 |
ABSTRACT |
In order to evaluate the diagnostic yield of a PCR assay for
patients with focal complications of brucellosis, we studied by PCR and
by conventional microbiological techniques 34 nonblood samples from 32 patients with different focal forms of brucellosis. The samples from
patients with brucellosis were paired to an equal number of control
samples from the same locations of patients whose illnesses had
different etiologies. Thirty-three of the 34 nonblood samples
(97%) from the brucellosis patients were positive by PCR, whereas
Brucella spp. were isolated from only 29.4% of the conventional cultures. For 11.4% of the patients, the confirmatory serological tests were either negative or showed titers below the
diagnostic range. Two patients (6.2%) from the control group, both
with tuberculous vertebral osteomyelitis, had a positive PCR result.
The brucella PCR of blood from these two patients was also positive,
and the two strains of Mycobacterium tuberculosis isolated were analyzed by the brucella PCR, with no evidence of amplification. These results show that the PCR assay is far more sensitive than conventional cultures, and this, coupled with its speed
and reduction in risk to laboratory workers, makes this technique a very useful tool for the diagnosis of focal complications of brucellosis.
| |
TEXT |
Brucellosis is a zoonosis
widely distributed around the world (8). In humans,
brucellosis behaves as a systemic infection with a very heterogeneous
clinical spectrum. The disease usually presents as fever with no
apparent focus, although in 20 to 40% of cases there are focal forms
(7). These focal forms of brucellosis have been described
in almost all organs and systems, with the osteoarticular and
genitourinary forms being more common and those affecting the heart and
the central nervous system being more severe (21).
The diagnosis of focal forms of brucellosis is sometimes difficult, as
the yield of conventional cultures of nonblood samples is as low as 10 to 40% among all cases (11, 14). Moreover, as
Brucella spp. are slowly growing pathogens, the cultures
require prolonged incubation, which can at times lead to excessive
delays in diagnosis. Furthermore, serological diagnosis lacks adequate specificity in areas where the disease is endemic, and the results of
serology for some slowly evolving focal forms are difficult to
interpret (2).
Our group has recently reported that the use of a PCR technique with
blood samples provides better results than conventional techniques in
the diagnosis of both primary infections and relapses of the disease
(15, 16). The aim of the study described here was to
evaluate the yield of this PCR technique with nonblood samples from
patients with different focal forms of brucellosis compared with that
of conventional microbiological techniques.
Patient population.
From January 1997 to December 1999, 34 nonblood samples from 32 patients with different focal complications of
brucellosis were studied by PCR assay. Samples came from synovial fluid
from eight patients; pus from abscesses from five patients; urine from five patients; cerebrospinal fluid (CSF) from five patients; vertebral or other bone tissue from four patients; sputum from two patients; renal cyst fluid from two patients; and pleural fluid, renal tissue, and thyroid tissue from one patient each. The two patients who had more than one focal form each provided two different samples. All
nonblood samples, excluding urine and sputum samples, were obtained by
conventional puncture, ultrasound, or computed tomography-guided puncture, depending on the site, or during open surgery.
The diagnosis of brucellosis was established by one of the following
criteria: first, isolation of Brucella spp. from blood or
any other body fluid or tissue sample or, second, the presence of a
compatible clinical picture together with the demonstration of specific
antibodies at significant titers or seroconversion. Significant titers
were considered to be a Wright's seroagglutination test titer of
1/160 or a Coombs antibrucella test titer of 1/320.
In all cases, cultures and PCR assays with the sample obtained from the
focus of infection, as well as a serological battery
including the rose
bengal plate agglutination test, Wright's seroagglutination
test, and
the Coombs antibrucella test, were carried out. For
28 patients
(87.5%) two or more sets of blood cultures were done,
and for 26 patients (81.25%) PCR was also done with a peripheral
blood
sample.
Blood cultures were processed in a BACTEC 9240 instrument
(Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) by
usual
techniques (
10), with incubation being maintained for
30 days and with blind subcultures performed on chocolate agar
and
brucella agar (Biomedics, San Sebastian de los Reyes, Madrid,
Spain)
after 10, 20, and 30 days. These subcultures were incubated
at 37°C
in a 5 to 10% CO
2 atmosphere for 7
days.
All nonblood samples were cultured onto blood and chocolate agar media,
MacConkey agar, and brucella agar. The samples of
urine, sputum, and
abscess pus were also inoculated onto a modified
Thayer-Martin medium
(Biomedics). The plates were incubated in
a 10%
CO
2 atmosphere at 37°C for at least 7 days. If
growth appeared,
the suspected colonies were identified by colonial
morphology;
Gram staining; oxidase, catalase, and urease tests; and
positive
agglutination with specific antiserum. All isolated strains
were
sent to the National Brucellosis Reference Laboratory in
Valladolid,
Spain, for definitive identification and biotyping. The
serological
tests were all performed by previously described techniques
(
1).
DNA extraction.
All samples destined for PCR study were
maintained at 
20°C until processing. The volume was varied
depending on the type of sample. Samples from the different tissues,
synovial fluid, and purulent sample collections were resuspended
in 1 ml of erythrocyte lysis solution (320 mM saccharose, 5 mM
MgCl2, 1% Triton X-100, 10 mM Tris HCl
[pH 7.5]), mixed, and centrifuged at 15,000 × g for
2 min. The supernatant was discarded, and the pellet was washed with
Milli-Q water and centrifuged as described above. This washing with
water was repeated until the pellet lost all reddish coloring. Purification and precipitation of DNA were performed as reported previously (16). DNA was extracted from urine and CSF
samples by boiling.
PCR assay.
Once the DNA was extracted, the amplification
process was performed by a previously described technique
(16). Briefly, this consisted of amplification of a 223-bp
fragment from the gene coding for the synthesis of an immunogenic
membrane protein of Brucella abortus BCSP31, described by
Mayfield et al. (13). This protein, with a molecular
mass of 31 kDa, is specific to the Brucella genus and
is present in all its biovars. The B4 and B5 primers described by Baily
et al. (3) were used in the amplification process.
Positive controls based on DNA from B. abortus B-19 were included in all the tests, as were negative controls, which contained all the elements of the reaction except DNA. A sample was considered positive when DNA with a molecular mass expected for the amplified product was fluorescent in the presence of ethidium bromide after 2%
agarose gel electrophoresis.
To guarantee the reliability of the results and detect any possible
contamination, all the samples were processed in duplicate.
The test
was considered positive if the signal from the amplified
fragment was
clearly visible in both samples. In order to study
the specificity of
the technique, all the samples from patients
with brucellosis were
paired to an equal number of samples from
controls with lesions at the
same site as the patients but with
a different etiology. The organisms
from the control group were
isolated from synovial fluid
(
Staphylococcus aureus from five
patients and
Neisseria meningitidis,
Neisseria gonorrhoeae,
and
Staphylococcus epidermidis from one patient
each), liver or splenic
abscesses (
Escherichia coli
from three patients and
Streptococcus intermedius and
Pseudomonas aeruginosa from one patient each),
urine
(
E. coli from three patients and
Klebsiella
pneumoniae and
Proteus mirabilis from one patient
each), CSF (
Streptococcus pneumoniae from two patients, and
N. meningitidis,
Mycobacterium tuberculosis,
and
S. epidermidis from one patient each), vertebral
osteomyelitis
tissue (
M. tuberculosis from three
patients and
S. aureus from
one patient), sputum (
S. pneumoniae and
Branhamella catarrhalis),
renal cyst
fluid (
P. mirabilis and
E. coli), pleural fluid
(
K. pneumoniae), renal tissue (
P. mirabilis), and
thyroid tissue (
Streptococcus agalactiae).
Statistical analysis.
Data were analyzed with the help of SPSS
8.0 for Windows. Sensitivity, specificity, and positive and negative
predictive values were calculated. Likelihood ratios and their 95%
confidence intervals were calculated with the Roc Curve Analyzer
program, version 6, as described by Centor (6).
Of the 32 patients included in the study, 26 (81.2%) were male
and 6 (18.7%) were female. The mean age of the group was 48.9
± 17.3 years (range, 19 to 81 years). For 17 patients (53.1%)
the
diagnosis of brucellosis was established by isolating the
pathogen in
blood cultures or in cultures of other samples. For
the remaining 15 patients (46.8%), the diagnosis was made by clinical-serological
means. All the strains isolated were identified as
Brucella
melitensis.
Relevant clinical data and the sites of the
different focal forms
are presented in Table
1.
Thirty-three of the 34 (97%) nonblood samples from the patients with
brucellosis had a positive PCR result, whereas
Brucella spp.
were isolated from cultures of the same samples from only
29.4% of the
patients. Clear visualization of the PCR-amplified
fragments was
possible for all positive samples (Fig.
1). The
only false-negative PCR result
corresponded to a sample of synovial
fluid for which culture was
positive. This sample was mistakenly
preserved with heparin.
Seventy-five percent of the patients whose
samples were culture
negative had previously received antibiotic
treatment for other
suspected diagnoses not related to brucellosis.
There was no difference
in the sensitivity of the PCR between
individuals in the treated group
and those who had not received
antimicrobial therapy prior to sample
extraction.
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|
FIG. 1.
Agarose gel electrophoresis and ethidium bromide
staining. Lane MW, DNA ladder (223 bp); lane 1, positive control
(B. abortus B-19 DNA); lane 2, a sample to which no DNA
was added; lane 3, synovial fluid from a brucellosis patient with knee
arthritis; lane 4, synovial fluid from a patient with knee arthritis
due to S. aureus; lane 5, urine sample from a patient
with orchiepididymitis due to B. melitensis; lane 6, urine sample from a patient with E. coli pyelonephritis;
lane 7, sample of pus from a liver abscess due to B.
melitensis; lane 8, sample of pus from a liver abscess due to
E. coli; lane 9, CSF from a brucellosis patient with
meningitis; lane 10, CSF from a patient with meningitis due to
M. tuberculosis. The photocomposition of the figure was
obtained from the original Polaroid films with a ScanJet IIcx scanner
(Hewlett-Packard, Corvallis, Oreg.) After the initial image was
scanned and saved as a tagged image file format file, the
file was opened in Adobe Photoshop (Adobe System, Inc., Seattle,
Wash.).
|
|
The rose bengal test was positive for 91.2% of the patients. Wright's
seroagglutination test and the Coombs antibrucella test
had titers
within the diagnostic range for 73.5 and 88.5% of the
patients,
respectively. Both confirmatory tests were negative
or showed titers
below the diagnostic range for 11.4% of
patients.
Two patients from the control group (6.2%), both with tuberculous
vertebral osteomyelitis, had positive PCR assay results.
The blood of
two patients, a 25-year-old shepherd with a 14-year
history of type I
diabetes mellitus and poor metabolic control
and a 38-year-old farmer,
also had positive PCR assay results.
The patients were treated
surgically and had favorable resolutions
of their infections
with conventional tuberculostatic therapy
plus doxycycline at 100 mg/12
h per os for 3 months. However,
neither of them demonstrated a
serological response by the rose
bengal test, the seroagglutination
test, or the Coombs antibrucella
test during the following 12 months.
In order to rule out a cross-reaction
with
Brucella spp.,
the two strains of
M. tuberculosis isolated
from these
patients were analyzed by the brucella PCR assay, and
both were
negative by the assay. Table
2 shows the
diagnostic
yields of the PCR versus those of the conventional cultures.
Almost one-third of all patients with brucellosis present with
focal complications, either initially or during the course
of the
disease (
7). The focal complications can affect any
organ
or system, which explains why these patients are not always
seen by
infectious disease specialists, so that many other medical
and surgical
specialists are involved in the care of these patients
(
17,
19).
Even when there is a high degree of suspicion, the diagnosis of
brucellosis is not always easy. Blood cultures lack sufficient
sensitivity; and serological tests, although more sensitive, are
not
wholly specific, may be difficult to interpret in areas where
the
disease is endemic, and may be negative during the early stages
of the
disease (
2,
20). Diagnosis of focal forms of brucellosis
can occasionally be difficult, not only because the physician
may not
consider it as the cause of the clinical picture but also
because even
when it is considered the yields of blood and nonblood
sample cultures
for patients with these forms are low (
7,
12,
14).
This is the first report to date that has analyzed the yield of a PCR
assay for the diagnosis of focal complications of human
brucellosis.
Our results show that the sensitivity of the PCR
assay was far superior
to that of conventional culture, which
was positive for only 29.4% of
cases. This low yield of culture
is similar to the 28.5% yield
reported by others for a series
of brucella meningitis or the 23.8%
reported for brucella spondylitis
(
12,
14). The
explanation for this low yield of conventional
culture appears to us to
be related more to the lack of suspicion
of brucellosis and the
widespread use of antimicrobial therapy
in patients with no etiological
diagnosis than to the technical
difficulty of isolating
Brucella spp. from clinical samples. In
the present study,
75% of patients whose cultures were negative
had received different
antibiotic treatments for various diagnostic
suspicions.
The high degree of sensitivity of the PCR assay, even for patients who
had previously received antibiotic therapy, seems to
be related to the
high degree of detection capacity of the technique.
We have estimated
in previous studies that the detection capacity
is about 10 to 100 fg
of DNA (
16). Such a small amount of DNA,
which is the
equivalent of that from just 2 to 20 cells, can be
expected to be
present in any clinical sample from a patient with
an active infection.
Moreover, PCR is able to amplify intramacrophage
pathogens or pathogens
which are damaged or nonviable as a result
of previous treatment and
which would be impossible to isolate
in conventional
cultures.
Despite the use of a wide battery of serological tests, 11.4% of the
patients with brucellosis in the present study failed
to show titers
within the diagnostic range. In fact, this figure
would have risen to
26.5% if we had used just the seroagglutination
test, which is the
serological test most commonly used by any
physician faced with a
possible diagnosis of brucellosis. The
low degree of sensitivity of the
seroagglutination test for the
focal forms of the disease is well known
and is due to the longer
duration of the clinical picture in these
patients. The immune
response to brucella infection is characterized by
an initial
rise in immunoglobulin M (IgM) agglutinating antibody titers
that
is followed within a few weeks by a switch to IgG antibodies,
with
the latter being detected better by other serological tests,
such as
the 2-mercaptoethanol agglutination test, the Coombs antibrucella
test,
an IgG enzyme-linked immunosorbent assay, or complement
fixation
(
2,
20).
The specificity of the PCR can be considered good, although the
existence of two patients with false-positive results requires
a search
for an explanation. Previous studies, as well as our
own results
obtained by PCR with the B4 and B5 primers, have demonstrated
the high
degree of specificity of the technique with a large panel
of
microorganisms. Only the DNA from
Ochrobactrum spp., a
pathogen
very closely related phylogenetically to
Brucella
spp., has been
amplified with these primers (
5).
Cross-reactivity with
M. tuberculosis has not been reported
before. The two patients with
false-positive results had habitual
contact with cattle, and the
suspensions of the pure cultures of these
strains of
M. tuberculosis failed to amplify. This led us to
think more of a coinfection
than of a false-positive result. In fact,
coinfection with
Mycobacterium and
Brucella has
been reported in our area, where the prevalence
of infections with both
of these organisms is high (
9). In
addition, in situ
immunohistochemical studies have demonstrated
that active effector
cells in granulomas from patients with tuberculosis
expressed high
levels of cytokines such as tumor necrosis factor
alpha, interleukin-6,
and gamma interferon, which might result
in activation of other latent
infections (
4). It is possible,
then, that severe
tuberculosis in these two patients led to the
subclinical reactivation
of a previous brucella
infection.
Finally, in addition to the high yield of the PCR assay for the
diagnosis of focal complications in patients with brucellosis,
other
important aspects of the technique make it especially attractive
for
use with patients with these types of complications. First,
PCR
is fast, providing results in 24 h, which is much less than
the
time required for conventional methods to rescue a fastidious
microorganism such as a
Brucella sp. Second, the technique
almost
completely obviates the necessity for direct handling of the
pathogen,
thus drastically reducing the risk of infection of laboratory
personnel (
18). Third, the sample can be stored at
20°C until
processing, thus enabling it to be collected by any
physician
and processed immediately or else stored and safely sent to
another
laboratory if
necessary.
In conclusion, the high degrees of sensitivity and specificity of the
PCR assay, together with its speed, versatility in sample
handling, and
risk reduction for laboratory personnel, make this
technique a very
useful tool for the diagnosis of focal complications
of
brucellosis.
| |
ACKNOWLEDGMENTS |
This work received financial support from the Inter-Ministerial
Commission for Science and Technology (CICYT) and the European Commission (grant IFD97-0539).
We thank Isolde Gornemann and Ian Johnstone for comments and help with
the English language version of the text.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unidad de
Enfermedades Infecciosas, Departamento de Medicina Interna, Hospital
Universitario Carlos Haya, C/Avda. Carlos Haya s/n, 29010 Malaga,
Spain. Phone: 34 952645809. Fax: 34 952645805. E-mail:
colmene{at}interbook.net.
| |
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Journal of Clinical Microbiology, October 2001, p. 3743-3746, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3743-3746.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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