Journal of Infection (2008) 57, 397e403

www.elsevierhealth.com/journals/jinf

Influence of brucellosis history on serological

diagnosis and evolution of patients with acute
brucellosis
Marı́a de los Ángeles Mantecón a, Marı́a Purificación Gutiérrez a,
Marı́a del Pilar Zarzosa b, Luis Fernández-Lago c, Juan de Dios Colmenero d,
Nieves Vizcaı́no c, Miguel Angel Bratos a, Ana Almaraz a, África Cubero a,
Maria Fe Muñoz a, A. Rodrı́guez Torres a, Antonio Orduña a,*

a
Unidad de Investigación, Hospital Clı´nico Universitario de Valladolid, Valladolid, Spain
b
Área Este de Atención Primaria SACYL, Valladolid, Spain
c
Dpto, Microbiologı´a y Genética Molecular, Universidad de Salamanca, Salamanca, Spain
d
Servicio de Enfermedades Infecciosas, Dpto de Medicina Interna, Hospital ‘‘Carlos Haya’’, Málaga, Spain

Accepted 19 August 2008

Available online 2 October 2008

KEYWORDS Summary Serological diagnosis of human brucellosis is problematic in endemic brucellosis

Brucellosis; regions and with patients having a history of brucellosis. The aim of this study is to ascertain
Serology; the serologic and evolutionary behavior of the tests of serum agglutination, Coombs anti-
Evolution; Brucella, immunocapture-agglutination, enzyme-linked immunosorbent assay (ELISA) IgG,
ELISA-IgG avidity; IgA, IgM and ELISA-IgG avidity against Brucella lipopolysaccharide (S-LPS), in patients with
Immunocapture- acute brucellosis based on whether or not a history of brucellosis exists. Titers and seroposi-
agglutination tivity in all the tests assayed were higher in the patients having brucellosis history (from
90.9% in ELISA-IgM to 100% in ELISA-IgG) than in the patients lacking such history (from
79.3% in ELISA-IgM to 86.2% in Coombs, immunocapture-agglutination, and ELISA-IgG). IgG
S-LPS avidity results in patients with brucellosis history were significantly higher (always over
84%) than in patients without brucellosis history (from 48.0% in the initial sera to 81% ten
months later) (p < 0.001). The titers of antibodies against Brucella in the initial sera and
ELISA-IgG avidity against S-LPS may allow distinguishing patients with brucellosis caused by
primary infection in the initial stages of the disease from patients seropositive due to prior
infections from Brucella.
ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Dpto Microbiologı́a, Facultad de Medicina, Avda Ramón y Cajal 7, 47005 Valladolid, Spain. Tel.: þ34 983423063;
fax: þ34 983423066.
E-mail address: orduna@med.uva.es (A. Orduña).

0163-4453/$34 ª 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinf.2008.08.005
398
Introduction
Brucellosis is a systemic disease produced by bacteria of
the genus Brucella, which affects humans and numerous
animal species. It is widely distributed throughout the
world.1 Human brucellosis is a bacteriemic process that
presents an undulating course, with a great tendency to-
wards relapses and evolving to a chronic state, and reinfec-
tions are frequent.2 The fact that the disease is greatly
polymorphic makes clinical diagnosis difficult. As a result,
confirmation by laboratory diagnosis is needed when bru-
cellosis is suspected.
Laboratory diagnosis of human brucellosis is based on
isolating Brucella by blood culture2,3 and on demonstrating
specific antibodies through serological tests.4e6 However,
the bacteria of the genus Brucella grow slowly and the per-
centage of hemoculture isolations varies according to the
clinical form of the brucellosis; the percentage is low in
chronic forms and those of prolonged evolution.3,7 In addi-
tion, Brucella bacteria are considered biological risk Group
III pathogens and manipulating them involves significant
contamination risks for the laboratory staff.8 For this rea-
son, most laboratories utilize serology for human brucello-
sis diagnosis.2,9 Serum agglutination (SAT)10 and Coombs
anti-Brucella, which detect mainly antibodies against Bru-
cella lipopolysaccharide, are among the serological tests
most often used for diagnosis of human brucellosis. How-
ever, interpreting the serological results from these tests
involves significant difficulties, as individuals who have suf-
fered prior Brucella infections can present specific anti-
bodies against Brucella that can last for long periods of
time.6 This makes it difficult to diagnose the infection in
patients with a history of this disease and those from zones
where it is endemic; in many cases it is hard to differenti-
ate between an active infection and an immune memory
from a previous infection.11 Similar difficulties appear in
diagnosing relapses, reinfections and chronic brucellosis,
when the serology presents patterns of secondary immune
response.9,12
Incorporating an enzyme-linked immunosorbent assay
(ELISA) to the diagnosis of human brucellosis13e16 has per-
mitted individualized studies of each class and subclass of
immunoglobulin. This has contributed to better knowledge
of each of them in the different phases and evolutionary
forms of the disease.6,15 However, in the majority of the
cases, even including ELISA in brucellosis diagnosis does
not clarify whether there is a reinfection or an immune
memory from past infection.
Avidity of specific IgG against the antibody increases
over time after the first immune system contact with this
Ag. The antibodies generated in secondary response to the
infection present high avidity for the antigen; this is used to
differentiate between primary and secondary infections in
several infectious diseases.17e19 Likewise, studying anti-
body avidity can be useful in distinguishing a recent infec-
tion from an immune memory, particularly in patients
with low or moderate antibody titers. However, experience
is not extensive in the case of brucellosis.20
An immunocapture-agglutination test (Brucellacapt) has
recently been introduced in the diagnosis of human bru-
cellosis,10 which has shown its usefulness in the diagnosis of
M. de los A. Mantecón et al.
this disease. However, how the test behaves in terms of the
existence or the absence of a history of Brucella infection is
not known.
The goal of our study is to ascertain the serological and
evolutionary behavior of the SAT, Coombs anti-Brucella,
Brucellacapt, and ELISA-IgG, IgA, and IgM tests against Bru-
cella smooth lipopolysaccharide (S-LPS) and against the
ELISA-IgG avidity test, based on whether a history of brucel-
losis exists or not. In addition, we aim to evaluate its possi-
ble diagnostic usefulness in both groups of patients.
Material and methods
Clinical samples
The serum sample was comprised of 258 sera from 51
patients diagnosed as having brucellosis. All the patients
presented a clinical picture of acute brucellosis, confirmed
by laboratory tests. A patient was considered to be a case
of acute brucellosis if he or she presented a clinical picture
compatible with the disease plus the fact that there had
been no clinical history of brucellosis during the year before
the current process; and
- Brucella was isolated from a pathological sample, or
- The first serum obtained had a serum agglutination titer
or a Coombs anti-Brucella test of 1/160, or
- A seroconversion or a fourfold rise in SAT titer was
observed.
Among the 51 patients, 22 had suffered from brucellosis
before the current brucellosis process (patients with
a history of brucellosis). During these 22 patients’ first
brucellosis process, all presented SAT titers above 1/640 at
some moment; 7 presented a positive blood culture and
Brucella melitensis biovar 3 was isolated in all 7 cases. All
these patients were cured, following complete treatment
guidelines, and they had not presented any clinical picture
compatible with brucellosis in the 12 months prior to the
current clinical picture of brucellosis (range 12e120
months). The remaining 29 patients had never suffered
from brucellosis before the present incidence. All the pa-
tients included in the study evolved favorably within less
than 3 months after the specific treatment, and none of
them presented a relapse or reinfection in the 12 months
after finalizing treatment.
All the patients gave a blood sample coinciding with the
medical consult in which they were diagnosed with a clinical
picture compatible with brucellosis (initial serum). The sera
obtained at 2, 4, 6, 8, and 10 months after initiating
treatment (evolution sera) were also studied.
As a negative control group, 412 sera were chosen at
random from healthy individuals living in areas in which
brucellosis is endemic (rural zones in the Region of Castilla
y León, Spain).
Methods
Blood samples were taken by veinopuncture. The serum
was separated from each sample and conserved divided in
aliquots at 20  C until used. For all patient and control
Influence of brucellosis history on serological diagnosis
sera, the presence of antibodies against Brucella spp was
determined by serum agglutination (SAT) (Linear Chemicals
S.L., Barcelona, Spain), Coombs anti-Brucella (Linear
Chemicals S.L., Barcelona, Spain), Brucellacapt (Vircell
SL, Granada, Spain),10 and ELISA specific IgG, IgM, and IgA
against S-LPS from B. melitensis 16M prepared in the labo-
ratory. The antibody avidity of IgG against Brucella S-LPS
was also studied using ELISA-IgG test.
Obtaining S-LPS antigen
S-LPS antigen was obtained from a Brucella broth (Becton
Dickinson, NJ, USA) culture of B. melitensis 16M (S) accord-
ing to the method described by Westphal and Jan (hot phe-
nol method),21 modified for Brucella.22
SAT and Coombs anti-Brucella test
Serum agglutination and Coombs test were performed in
tube by the double-dilution method, with an initial dilution
of 1/20, using the commercial antigen Brucella abortus
(Linear Chemicals, Barcelona, Spain). Serum agglutination
reactions were read 24 h after incubation at 37  C. The
highest serum dilution showing >50% agglutination was con-
sidered the agglutination titer. Coombs test4 was per-
formed with SAT tubes, washing 3 times with phosphate
buffered saline (PBS) pH 7.2. After the last wash, the sedi-
ment was resuspended in 1 ml PBS and 0.05 ml of previously
standardized anti-total human immunoglobulin (Sanofi Pas-
teur, France) was added to each tube. The tubes were
shaken and then incubated at 37  C for 24 h. Readings
were performed in the same way as for the SAT test.
Immunocapture-agglutination test (Brucellacapt@)
(Vircell SL, Granada, Spain)
This test was performed as specified by the manufacturer.
Briefly, 50 ml samples of each serum dilution were added to
the wells of a U-bottom microplate coated with an antihu-
man immunoglobulin. Next, 50 ml of the antigen suspension
(colored B. melitensis, killed by formaldehyde treatment)
was added. The plates were sealed with adhesive tape
and incubated at 37  C for 24 h in a dark humid chamber.
The microplates were then read, those showing agglutina-
tion over the well bottom being considered positive.
The positivity threshold of the agglutination-based tests
was determined by means of diagnostic efficiency curves
using the statistical program SPSS v. 11.5. Positives were
considered to be a SAT titer of 1/40, a titer of 1/80 for the
Coombs tests, and 1/160 for Brucellacapt.10,23
ELISA-IgG, IgA and IgM S-LPS tests
Briefly, for performing the ELISA techniques, we added
100 ml of the S-LPS solutions in PBS (Oxoid, England) to each
microplate well at the concentration of 5 mg/ml previously
established in standardization. The microplates were incu-
bated at 4  C for 24 h. Once the antigen was set, the plates
were washed with PBS and blocked with a solution of PBS-T-
BSA (PBS with 0.05% Tween 20 and Fraction V of bovine
seroalbumin (BSA) (Sigma) at 1%) for an hour at room
temperature. They were then washed with PBS-T and
100 ml of problem serum, or positive or negative control
serum, diluted to 1/100 in PBS-T-BSA was added to each
of the wells. All the sera were double-assayed. The plate
was incubated in a double-boiler at 37  C for 1 h and, after
399
5 washes in PBS-T, 100 ml of horseradish peroxidase conju-
gated rabbit antibodies against human anti-IgG (Gamma
chains), IgM (Mu chains) or IgA (Alpha chains) (DAKO, Den-
mark) was added. Following a new incubation at 37  C for
30 min and new washes, development was performed with
orthophenyldiamine (Sigma). Positivity threshold was es-
tablished as the average plus 2 standard deviations of the
absorbance from the 412 control group sera. Absorbance
values higher than the threshold value were considered
positive, and those under it, negative.
IgG avidity against S-LPS
For standardization of the test of IgG avidity against S-LPS
of B. melitensis 16M, we used the initial sera from 10 pa-
tients suffering from acute brucellosis without any history
of brucellosis and 10 sera from the same patients obtained
10 months after initiating treatment, utilizing the optimum
conditions obtained in the ELISA-IgG S-LPS standardization.
To do so, the S-LPS-coated wells were incubated with the
sera (6 wells for each serum sample); subsequently, half
the wells (3 per serum) were washed with different urea
concentrations (4, 6, and 8 M) in PBS-T-BSA, leaving the
urea solution to act for 3 or 5 min. The remaining 3 wells
for each serum were washed with PBS-T-BSA alone. The
conditions that established greater absorbance differences
between the urea-treated sera and the sera not treated
with urea were utilized to perform the test (8 M urea con-
centration in PBS-T-BSA and 3-min incubation at room tem-
perature). All the sera from the same patient were assayed
in the same microplate. The results were expressed as per-
cent of decrease in the absorbance of the urea-treated sera
with respect to the same sera without urea treatment.
Evolution studies
For the studies on the evolution of the SAT, Coombs, and
Brucellacapt tests, the percent of variation of the loga-
rithm of the inverse of the titer of each evolution serum in
relation to that of the corresponding initial sera was
calculated. To determine the antibody levels of each serum
against each antigen in the ELISA tests, a standard curve
was performed with different dilutions of a mixture of sera
from brucellosis patients strongly positive against Brucella.
A value of 1000 units/ml was assigned to this serum mix-
ture. This standard curve was included in the ELISA plates,
and the serum antibody concentrations of each plate were
determined by comparison with the standard curve. Each
point of the curves represented the average of the percent
of variation in the evolution serum titers with respect to
the result obtained from the initial sera.
Statistical analysis
The comparison between the avidity of the sera obtained at
each evolution moment for patients with brucellosis history
and for patients not having any brucellosis history was
performed with the non-parametric ManneWhitney U test
(SPSS v. 11.5). Differences of p  0.05 were considered sta-
tistically significant.
To compare positivity percents between the patients
with and without brucellosis history, a confidence interval of
95% was calculated for the difference within each group.
This difference was considered significant when the interval
did not contain zero.24 Analysis of variance (ANOVA) was
400 M. de los A. Mantecón et al.

used to compare whether the variation of avidity in each pa-
tient group during the period of time studied was significant.
Results
Patient characteristics
Fifty-one patients with acute brucellosis (22 having clinical
histories of prior brucellosis and 29 without such a history)
were included in the study; 10 were women and 41 were
men (Table 1). The average age of patients lacking brucel-
losis history was 37.9  17.5 years (range, 7e71 years) and
that of patients with this history was 44.3  16.8 years
(range, 27e62). Blood cultures were positive in 16 cases
(58.6%) in the patient group without clinical brucellosis his-
tory and in 12 cases (54.5%) in the group having clinical bru-
cellosis history. B. melitensis biovar 3 was isolated in all
cases. In the remaining cases in which the blood culture
was negative, the patients were diagnosed on the basis of
the clinical findings and serological criteria.
patients lacking brucellosis history. All the patients suffer-
ing from acute brucellosis who had a history of brucellosis
presented titers equal to or above 1/320 in the Coombs
test, while 41.2% of the patients lacking previous history of
brucellosis presented titers equal to or below 1/160 in this
test. All the serological tests studied (SAT, Coombs anti-
Brucella test, Brucellacapt, and ELISA-IgG, IgM, and IgA
against S-LPS) showed a higher positivity percent in
patients with brucellosis history, although no statistically
significant differences were observed between both groups
(Table 2). In the control sera collected from healthy
patients in the brucellosis endemic region where this study
was conducted, 2 sera of 412 sera (0.48%) were SAT positive
(both presented a titer of 1/20), 5 were Coombs test posi-
tive (1.2%) (1 serum presented a titer of 1/320 and the
remaining 4 had titers lower than 1/160), and 15 (3.6%)
were Brucellacapt positive (1 serum 1/2560, 2 sera 1/640,
1 serum 1/320 and 11 sera 1/160).
Antibody evolution based on existence
or absence of brucellosis history

Diagnostic results When antibody evolution was studied on the basis of

We distributed the patients into two groups based on
whether brucellosis history existed or not and we compared
the titers and the seropositivity in the diagnostic sera of
each patient group detected by each technique. The
patients having brucellosis history generally presented
higher levels of antibody titers in the initial sera than
whether brucellosis history existed or not, the evolution
curves of SAT, Coombs anti-Brucella, and Brucellacapt were
similar; the average of the antibody titers of patients hav-
ing a history of brucellosis was higher than that of those
without such a history. The antibody levels of patients for
whom a history existed remained fairly constant, values
near the initial serum values being maintained throughout

Table 1 Characteristics of patients with acute brucellosis included in the study distributed according to the presence or the
absence of a history of brucellosis
Without history (no. 29) With history (no. 22)
Gender Women e 6 (20.6%) Women e 4 (18.1%)
Men e 23 (79.3%) Men e 18 (81.8%)
Age (mean  SD) 37.9  17.5 44.3  16.8
Epidemiological characteristics
Livestock or crop farmer 14 (48.2%) 13 (59.1%)
Veterinarian 3 (10.3%) 2 (9.0%)
Rural home 23 (79.3%) 21 (95.4%)
Accidental contact 4 (13.7%) 1 (4.5%)
Unknown origin 2 (6.8%) 0
Clinical characteristics
High fever (>38  C) 27 (93.1%) 21 (95.4%)
Chills 22 (75.8%) 16 (72.7%)
Sweating 26 (89.6%) 18 (81.8%)
Asthenia 24 (82.7%) 18 (81.8%)
Headache 17 (58.6%) 13 (59.0%)
Anorexia 17 (58.6%) 14 (63.6%)
Weight loss 12 (41.3%) 10 (45.4%)
Cough 5 (17.2%) 5 (22.7%)
Hepatomegaly 17 (58.6%) 10 (45.4%)
Splenomegaly 13 (44.8%) 8 (36.3%)
Orchitis 4 (13.7%) 3 (13.6%)
Sacroiliitis 5 (17.2%) 4 (18.1%)
Arthritis 1 (3.4%) 1 (4.5%)
Influence of brucellosis history on serological diagnosis 401

was a progressive increase in avidity as the time from

Table 2 Percent of positives based on the presence or ab-
sence of a history of brucellosis
With Without Proportion (%)
history (%) history (%) difference
(n Z 22) (n Z 29) (CI 95%)
SAT 95.5 82.8 12.7 (7.6 to 33.0)
Coombs test 100 86.2 13.8 (2.8 to 30.3)
Brucellacapt 95.5 86.2 9.3 (100.0 to 28.5)
IgG-ELISA 100 86.2 13.8 (2.8 to 30.3)
IgM-ELISA 90.9 79.3 11.6 (11.4 to 34.6)
IgA-ELISA 90.9 86.2 4.7 (16.7 to 26.1)
SAT: seroagglutination test. ELISA assays using smooth lipopoly-
saccharide of Brucella melitensis 16M as antigen. CI: confidence
interval.
the study period. In contrast, the average antibody titers
in patients not having a brucellosis history decreased
much more rapidly; at six months’ evolution, the values
were 40% lower than the initial sera results. Comparing
both groups, the antibody levels of the patient group
with brucellosis history remained more elevated than
those of the patient group without brucellosis history;
this difference was statistically significant (SAT: month 2:
p Z 0.038, month 4: p Z 0.042, month 6: p Z 0.030, and
month 8: p Z 0.025; Coombs test: month 2: p < 0.001,
month 4: p Z 0.010, month 6: p Z 0.004, and month 10:
p Z 0.028; and Brucellacapt: month 2: p Z 0.004, month
4: p Z 0.042, and month 6: p Z 0.004).
With the ELISA technique, the behavior of the three
types of immunoglobulin against S-LPS was similar in the
two patient groups. In IgM and IgA ELISA tests, the evolution
curves of patients having brucellosis history presented
antibody titers higher than those of the patients lacking
this history, although no statistically significant differences
were observed, except for the ELISA-IgM test in the 2nd and
6th months (p Z 0.008 and p Z 0.002, respectively). In con-
trast, in the ELISA-IgG test, the evolution curve of the anti-
body titers was higher in patients not having a history,
although the differences were not statistically significant.
Avidity evolution of IgG antibodies in patients
with and without brucellosis history
The evolution of IgG avidity was studied for 10 months from
the moment of diagnosis in patients with and without
brucellosis history. In patients not having a history, there
diagnosis lengthened (from 54.5%  12 in the initial serum
to 76.7% at the tenth month) (Table 3). In contrast, in pa-
tients having brucellosis history, avidity remained at values
slightly above 90% over the entire ten months studied.
Throughout this period, IgG avidity was significantly higher
(p < 0.0001) in patients having brucellosis history than in
those without such a history.
Discussion
Serologic response in human brucellosis is characterized by
the persistence of antibodies against S-LPS; such antibodies
can stay elevated for even years after the disease has been
cured.6 On the other hand, in endemic brucellosis areas,
the prevalence of antibodies against Brucella can be high,
there being patients with elevated titers of antibodies
against Brucella. In our study, 3.6% of control group sera
from the healthy population were positive in Brucellacapt
test, 1.2% in Coombs test, and 0.48% in SAT test. This prev-
alence of Brucella antibodies makes serological diagnosis
difficult for brucellosis patients from these areas.2 Klerk
and Anderson’s studies14 show that patients exposed to or
with a prior history of the disease have a basal antibody
level higher than that of patients without such a history.
Similarly, in our study we found in the initial sera that pa-
tients with brucellosis history presented SAT, Coombs
anti-Brucella and Brucellacapt test antibody titers that
were more elevated than those of patients without such
a history. Similar results were found in the tests of ELISA-
IgG, ELISA-IgA and IgM against S-LPS.
In the SAT, Coombs and Brucellacapt tests, the antibody
evolution curves in patients having brucellosis history all
presented higher titers than those of patients lacking this
history, the differences being statistically significant. In
addition, the antibody levels in patients with brucellosis
history stayed elevated for a longer period, as a result of
the memory effect of the immune response.25,26
Studying the immune response for each group based on
immunoglobulin class revealed that IgG antibodies against
S-LPS evolved similarly in both groups, whether or not
brucellosis history existed; both groups maintained high
levels, close to those of the initial sera. This behavior has
been observed by other authors (i.e. Ariza et al.6 and Baldi
et al.27), who reported a long persistence of IgG antibodies
against S-LPS at elevated titers for over six months, inde-
pendently of whether the case was a relapse, reinfection
or primary infection.12

Table 3 Percent of IgG avidity of specific IgG antibodies in patients with and without a previous history of brucellosis
Months of evolution Without history % (CI 95%) With history % (CI 95%) Statistical significance
Month 0 54 (48.2e59.7) 94.0 (87.4e100.6) p Z 0.0000
Month 2 59.6 (51.5e63.5) 94.9 (87.9e101.8) p Z 0.0000
Month 4 68.2 (61.8e74.5) 91.8 (84.5e99.1) p Z 0.0001
Month 6 72.7 (69.6e75.8) 93.6 (90.0e97.2) p Z 0.0000
Month 8 73.9 (71.0e76.7) 93.7 (90.4e96.9) p Z 0.0000
Month 10 76.7 (74.5e78.9) 92.3 (89.7e94.8) p Z 0.0000
Statistical significance of variation of aviditya p Z 0.000 p Z 0.977
a
ANOVA (analysis of variance).
402
IgG avidity in the patient group lacking brucellosis
history showed a progressive increase, while the avidity
percent in those having this history remained constant and
above 84%. The differences in avidity obtained for both
groups were significant. The differences observed show
that in brucellosis, IgG avidity is low in the first contact
with the antigen and increases as the time of evolution of
the disease progresses, as has been observed in other
diseases.17e20 Clinical diagnosis of brucellosis is frequently
difficult because of the polymorphism of the disease. In ad-
dition, bearing in mind that most brucellosis patients who
suffer a reinfection present high antibody titers, problems
arise in patients suspected of suffering from brucellosis
who present low or moderate antibody titers that may be
due to a recent infection or an old immunological mem-
ory.9,12 In these cases, the IgG avidity test can be useful,
as elevated IgG avidity in these patients would be indica-
tive of immune memory, not current infection. In contrast,
patients with titers at the diagnostic borderline and low
avidity would be indicative of primary infections. Likewise,
patients with a history of Malta fever suffering from a new
brucellosis process will present high antibody titers to-
gether with high IgG avidity anti-S-LPS against Brucella.
In summary, we can conclude that antibody levels in
patients having brucellosis history remain higher and for
a longer period of time than in patients without this history,
except for ELISA-IgG against S-LPS. IgG evolution is in-
dependent of the presence or the absence of brucellosis
history, since it remains elevated in both patient groups
over the period studied. ELISA-IgG avidity against S-LPS
allows us to diagnose brucellosis in endemic areas in
patients who present low or moderate antibody titers,
distinguishing patients with brucellosis from primary in-
fection in the initial stages of the disease from patients
seropositive due to prior infections from Brucella.
Acknowledgements
This study was performed with the financial support of the
Health Board of the Junta de Castilla y León (Brucellosis
Research Program), funding from the Junta de Castilla y
León (Ref. VA062A06), and a scholarship from the Thematic
Network (Red Temática) on Brucellosis Research at the
Health Institute Carlos III (Ref. G03-204 and FIS 05/2632).
We wish to thank Prof. C. Nolan for collaborating with revis-
ing the English version. The experiments in this study com-
ply with current Spanish laws.
References
1. Boschiroli ML, Foulongne V, ÓCallaghan D. Brucellosis: a world-
wide zoonosis. Curr Opin Microbiol 2001;4:58e64.
2. Young EJ. Brucella species. In: Mandell GL, Bennett JE, Dolin R,
editors. Mandell, Douglas, and Bennett’s principles & practice
of infectious diseases. 6th ed. New York: Churchill Livingstone;
2005. p. 2669e74.
3. Yagupsky P. Detection of Brucellae in blood cultures. J Clin
Microbiol 1999;37:3437e42.
4. Hall WH, Manion RE. Comparison of the Coombs test with other
methods for Brucella agglutinins in human serum. J Clin Invest
1953;32(1):96e106.
M. de los A. Mantecón et al.
5. Dı́az R, Moriyón I. Laboratory techniques in the diagnosis of hu-
man brucellosis. In: Young EJ, Corbel ML, editors. Brucellosis:
clinical and laboratory aspects. New York: CRC Press, Inc.;
1989. p. 73e83.
6. Ariza J, Pellicer T, Pallares R, Foz A, Gudiol F. Specific anti-
body profile in human brucellosis. Clin Infect Dis 1992;14(1):
131e40.
7. Gotuzzo E, Carrillo C, Guerra J, Llosa L. An evaluation of
diagnostic methods for brucellosis: the values of bone marrow
culture. J Infect Dis 1986;153:122e5.
8. Yagupsky P, Peled N, Riesenberg K, Banai M. Exposure of hospi-
tal personnel to Brucella melitensis and occurrence of labora-
tory-acquired disease in an endemic area. Scand J Infect Dis
2000;32:31e5.
9. Ariza J. Brucellosis. Curr Opin Infect Dis 1996;9:126e31.
10. Orduña A, Almaraz A, Prado A, Gutiérrez MP, Garcı́a-Pascual A,
Dueñas A, et al. Evaluation of an immunocapture-agglutination
test (Brucellacapt) for serodiagnosis of human brucellosis.
J Clin Microbiol 2000;38:4000e5.
11. Martı́n Moreno S, Guinea-Esquerdo L, Carrero González P. El
diagnóstico de la Brucelosis en un área endémica. Valoración de
las pruebas diagnósticas habituales. Med Clı´n (Barna) 1992;98:
481e5.
12. Pellicer T, Ariza J, Foz A, Pallarés R, Gudiol F. Specific anti-
bodies detected during relapse of human brucellosis. J Infect
Dis 1988;157:918e24.
13. Saz JV, Beltran M, Diaz A, Agulla A, Merino FJ, Villasante PA,
et al. Enzyme-linked immunosorbent assay for diagnosis of bru-
cellosis. Eur J Clin Microbiol Infect Dis 1987;6(1):71e4.
14. de Klerk E, Anderson R. Comparative evaluation of the
enzyme-linked immunosorbent assay in the laboratory diagno-
sis of brucellosis. J Clin Microbiol 1985;21:381e6.
15. Araj GF, Lulu AR, Mustafa MY, Khateeb MI. Evaluation of ELISA
in the diagnosis of acute and chronic brucellosis in human be-
ings. J Hyg 1986;97:457e69.
16. Colmenero JD, Porras J, Cardenas A, Ocon P, Reguera JM,
Delgado M, et al. Evaluación de la prueba de EIA Chromotitre
en el diagnostico de la brucelosis humana. Enferm Infecc
Microbiol Clin 1994;12(2):60e5.
17. Polanec J, Seppälä I, Rousseau S. Evaluation of protein e dena-
turating immunoassays for avidity of immunoglobulin G to Ru-
bella virus. J Clin Lab Anal 1994;8:16e21.
18. Grangeot-Keros L, Mayaux MJ, Lebon P. Value of Cytomegalovi-
rus (CMV) IgG avidity index for the diagnosis of primary CMV
infection in pregnant women. J Infect Dis 1997;175:944e6.
19. Lazzarotto T, Spezzacatena P, Pradelli P, Abate DA, Varani S,
Landini MP. Avidity of immunoglobulin G directed against hu-
man cytomegalovirus during primary and secondary infections
in immunocompetent and immunocompromised subjects. Clin
Diagn Lab Immunol 1997;4(4):469e73.
20. Gutiérrez FJ, Rodrı́guez VM, Maroto MC. Relación entre el
tiempo de evolución de la brucelosis humana y la avidez de
anticuerpos IgG especı́ficos. Rev Med Chil 1995;123:819e22.
21. Westphal O, Jann K. Bacterial lipopolysaccharides: extraction
with phenolewater and further applications of the procedure.
In: Whistler RL, editor. Methods in carbohydrate chemistry,
vol. 5. New York: Academic Press; 1965. p. 83e91.
22. Redfearn MS. An immunochemical study of antigens of
Brucella extracted by the Westphal Technique. Madison,
USA: University of Wisconsin; 1960.
23. Mantecon MA, Gutierrez P, Zarzosa MP, Dueñas AI, Solera J,
Fernández-Lago L, et al. Utility of an immunocapture-ag-
glutination test and an enzyme-linked immunosorbent assay
test against cytosolic proteins from Brucella melitensis
B115 in the diagnosis and follow-up of human acute brucel-
losis. Diagn Microbiol Infect Dis 2006;55:27e35.
24. Altman D, Bryant T, Gardner MJ, Machin D. Statistics with con-
fidence. London: British Medical Journal Books; 2000.
Influence of brucellosis history on serological diagnosis
25. White RG. Immunoglobulin profiles of the chronic antibody re-
sponse: discussion in the relation to brucellosis infections.
Postgrad Med J 1978;54:595e602.
26. Gazapo E, González Lahoz J, Subiza JL. Changes in IgM and IgG
antibody concentrations in brucellosis over time: importance
for diagnosis and follow up. J Infect Dis 1989;159:219e25.
403
27. Baldi PC, Giambartolomei GH, Goldbaum FA, Abdón LF,
Velikovsky CA, Kittelberger R, et al. Humoral immune re-
sponse against lipopolysaccharide and cytoplasmic proteins
of Brucella abortus in cattle vaccinated with B. abortus
S19 or experimentally infected with Yersinia enterocolitica
serotype O:9. Clin Diagn Lab Immunol 1996;3:472e6.