0095-1137/04/$08.00
⫹
0 DOI: 10.1128/JCM.42.10.4480–4486.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Detection of
Paracoccidioides brasiliensis
gp70 Circulating Antigen
and Follow-Up of Patients Undergoing Antimycotic Therapy
Silvia Helena Marques da Silva,
1Daniela de Mattos Grosso,
1Jose´ Daniel Lopes,
1Arnaldo Lopes Colombo,
2Maria Heloisa Souza Lima Blotta,
3Fla´vio Queiroz-Telles,
4and Zoilo Pires de Camargo
1*
Department of Microbiology, Immunology and Parasitology
1and Department of Infectious Disease,
2Federal University of
Sa
˜o Paulo, Sa
˜o Paulo, and Department of Clinical Pathology, Medical School, State University of Campinas,
Campinas,
3Sa
˜o Paulo, and Department of Community Health, Medical School,
Federal University of Parana
´, Curitiba, Parana
´,
4Brazil
Received 22 March 2004/Returned for modification 5 April 2004/Accepted 18 June 2004
Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Central and South America,
is caused by the dimorphic fungus
Paracoccidioides brasiliensis
and has a high prevalence in Brazil.
Glyco-proteins of 43 and 70 kDa are the main antigenic compounds of
P. brasiliensis
and are recognized by Western
blotting by 100 and 96% of PCM patient sera, respectively. In the present study, an inhibition enzyme-linked
immunosorbent assay (ELISA) was used to detect gp70 in different biological samples from patients with PCM.
gp70 was detected in 98.76% of 81 serum samples, with an average concentration of 8.19
g/ml. The test was
positive for 100% of the patients with the acute and chronic unifocal forms of PCM and 98.43% of the patients
with the multifocal chronic form, with average concentrations of 11.86, 4.83, and 7.87
g/ml, respectively.
Bronchoalveolar lavage fluid from 23 patients with pulmonary unifocal PCM and 14 samples of cerebrospinal
fluid from patients with neurological PCM were also tested for gp70 detection, with the test showing 100%
sensitivity and 100% specificity, with mean gp70 concentrations of 7.5 and 6.78
g/ml, respectively. To
investigate the potential of gp70 detection by inhibition ELISA for the follow-up of PCM patients during
antimycotic therapy with itraconazole (ITZ), the sera of 23 patients presenting with the chronic multifocal form
of PCM were monitored at regular intervals of 1 month for 12 months. The results showed a decrease in
circulating gp70 levels during treatment which paralleled the reduction in anti-
P. brasiliensis
antibody levels.
The detection of
P. brasiliensis
gp70 from the biological fluids of patients suspected of having PCM proved to
be a promising method for diagnosing infection and evaluating the efficacy of ITZ treatment.
Paracoccidioidomycosis (PCM), the most prevalent systemic
fungal mycosis in Latin America, is caused by
Paracoccidioides
brasiliensis
, a thermally dimorphic fungus (5, 34). Different
clinical forms of the disease result from the interaction of
different
P. brasiliensis
strains with the host, a phenomenon
that may be related to the virulence of the infecting strain.
Some investigators (21, 37, 38, 41) believe that the mechanism
of virulence is determined by chemical components present on
the fungal wall, but others contest this explanation (44).
Diag-nosis of PCM is usually based on the demonstration of
mul-tibudding yeast cells in different biological specimens or the
detection of specific antibodies by histopathology or culture.
During the past decade, substantial progress has been made in
the development of innovative approaches and methods for
the serological diagnosis of PCM. Clinically relevant antigens
have been identified and adapted for use in immunoassays for
the detection of specific antibodies (12). Some investigators
have tried to detect circulating antigen in PCM patients using
polyvalent antigens or antibodies in different assays such as
competition enzyme-linked immunosorbent assay (ELISA)
(16), immunoradiometric assay (13),
immunoelectrophoresis-immunodiffusion (17), counterimmunoelectrophoresis (35),
passive hemagglutination inhibition (24), inverted linear
im-munoelectrophoresis (23), and immunoblotting (28); but all of
these assays presented low sensitivities. Go
´mez et al. (19) were
the first to use monoclonal antibodies (MAbs) to detect the
87-kDa circulating antigen in PCM patients by inhibition
ELISA (inh-ELISA), which had 80.4% sensitivity. More
re-cently, the same technique was used to detect gp43 in serum,
cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL)
fluid of PCM patients with 95.1% sensitivity and 100%
speci-ficity (25) and to monitor PCM patients during azole treatment
(26).
The gp43 glycoprotein from
P. brasiliensis
is the main
anti-gen used for the diagnosis of PCM (32, 33, 40), being
recog-nized by virtually all sera from infected patients by different
serological methods (25, 27). Another important glycoprotein
expressed by the fungus is gp70, recognized by 96% of PCM
patient sera (3). gp70 is predominantly composed of
polysac-charides (27) and is able to induce proliferation of lymphocytes
from PCM patients (2). gp70 is also detected in the urine of
patients with the acute form of PCM (36). The treatment of
mice by injection of anti-gp70 MAb abolishes lung infection,
suggesting that gp70 may facilitate fungal installation and the
progression of lesions during the primary infection (27).
Detection of circulating antigens in patients with PCM has
proved to be useful not only for diagnostic purposes but also as
a tool to evaluate clearance of the fungal burden during
treat-ment. Go
´mez et al. (19) reported on the use of the inh-ELISA
* Corresponding author. Mailing address: Universidade Federal de
Sa˜o Paulo (UNIFESP), Disciplina de Biologia Celular, 04023-062, Rua
Botucatu, 862, 8° andar, Sa˜o Paulo, SP, Brazil. Phone: 55 11 5576 45
23. Fax: 55 11 5571 58 77. E-mail: zoilo@ecb.epm.br.
to detect gp87 circulating antigen in sera from patients with
active disease and in patients receiving antimycotic therapy
(20). A previous study that used the same methodology was
able to detect gp43 in 96.29% of PCM patients, mainly in those
with the acute form of the disease (100%) (25). Moreover,
patients cleared this antigen during itraconazole (ITZ)
ther-apy, suggesting that this molecule may be used to assess the
host response to antifungal therapy.
When it is considered that, in addition to gp43, gp70 is an
important molecule in the
P. brasiliensis
system, in the present
study we investigated the application of gp70 antigen detection
in different biological specimens to obtain a diagnosis of PCM
as well as to assess antigen clearance during antifungal
treat-ment for disseminated disease.
MATERIALS AND METHODS
Clinical samples for PCM diagnosis.Blood specimens from a total of 81 patients with active PCM were taken at the time of the primary diagnosis. The diagnosis of PCM was established for all patients by direct KOH examination, isolation or the fungus by culture, and/or conventional positive serological tests. Samples were collected from patients at different times between March 1990 and December 2001. The samples were from patients seen at Hospital Sa˜o Paulo, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil; Hospital das Clínicas, Fed-eral University of Parana´, Curitiba, Brazil; and Hospital das Clínicas, University of Campinas, Campinas, Brazil. The 81 patients tested (73 males and 8 females) were classified by their clinical presentations, as follows: 11 had the acute form of PCM (mean age, 18.8 years) and 70 had the chronic form (mean age, 46.7 years), including 64 patients with multifocal disease and 6 with unifocal disease. Biological material from 14 patients with neurological PCM (neuro-PCM) were also tested, including 14 CSF samples and 11 serum samples, all of which were obtained before treatment. In addition, 13 BAL fluid samples from patients with pulmonary PCM were tested.
Serum samples from patients (n⫽71) with other mycologically and/or sero-logically confirmed mycoses (51 serum samples from individuals with active histoplasmosis and 20 individuals with cryptococcosis), obtained at the time of diagnosis, were also evaluated. Ninety-three serum samples from healthy volun-teers (blood donors), 6 CSF samples from patients with no fungal disease, and 10 BAL fluid samples from individuals with tuberculosis were included as negative controls.
Clinical samples for monitoring of therapy.In order to evaluate the decrease in the antigen concentration in response to antifungal therapy, a total of 23 patients with active PCM were evaluated. Sequential serum samples were ob-tained from all selected patients, including one sample collected at the time of diagnosis (baseline) and at least seven others collected during 8 to 12 months of treatment. Samples were collected from patients attending the Infectious Dis-eases Division, Hospital das Clínicas, Federal University of Parana´, between 2001 and 2002. The patients were males (mean age, 47.3 years) with the chronic multifocal form of the disease who were successfully treated with ITZ. The good clinical response to antifungal therapy exhibited by all selected patients was characterized by the cessation of all clinical signs and symptoms related to the infection, significant improvement in the chest X-ray pattern of pulmonary le-sions, the absence of fungi in biological specimens, and negative serology or the presence of low titers of specific antibodies (as determined by the immunodif-fusion [ID] test).
Fungal isolates, antigen preparations, and gp70 purification.P. brasiliensis
113 (which was originally from the Faculdade de Medicina da Universidade de Sa˜o Paulo and which was a gift of C. S. Lacaz) was transformed to the yeast phase, and exoantigen was produced as described by Camargo et al. (7).P.
braziliensis113 was selected, as previous studies have shown that it produces large amounts of gp70. The yeast cells were harvested from this growth, washed three times with lysing buffer (0.05 mM Tris-HCl, 5 mM EDTA), and resus-pended in 50 ml of the same buffer. Yeast cells were broken with glass beads (diameter, 425 to 600m; Sigma) in a vortex mixer. A mixture of protease inhibitors containing 1 mM phenylmethylsulfonyl fluoride (Sigma) and 1 mM EDTA was added four times during the process. The homogenate was then centrifuged at 13,000⫻gfor 30 min at 4°C. The supernatant (which contained the cytoplasmic antigen) was recovered and used for gp70 purification. The cytoplasmic yeast antigen was fractionated by gel filtration chromatography with Sephadex G-50 resin (Pharmacia). The eluted fractions containing the gp70
molecule were concentrated and further purified by gel filtration on a HiTrap S-200 column (Pharmacia) to eliminate high-molecular-mass contaminants. The protein concentration was determined by the method of Bradford (4). To verify the real purification of gp70, the material was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), silver staining, and immunoblotting assays.
MAb production and immunization protocols.In the first schedule of immu-nization, 6-week-old BALB/c mice were injected intraperitoneally with a mac-erated polyacrylamide gel containing gp70 (gp70 was cut from the SDS-poly-acrylamide gel) at 2-week intervals for 4 months. In a second schedule, mice were immunized subcutaneously with 50g of gp70 (purified through MAbs obtained in the first fusion) in phosphate-buffered saline (PBS) every 2 weeks. gp70 was incorporated in Freund’s complete adjuvant in the first injection and in incom-plete Freund’s adjuvant in the subsequent ones. Injections were always made at four different sites in the axillary and inguinal regions with a final volume of 100 l per site. Before each immunization, the mice were bled through the ocular plexus, and the serum was separated by centrifugation and stored at⫺20°C. The final immunization was made intravenously 2 days before cell fusion.
Fusion protocols.Cells of murine myeloma line sp2 were fused with spleen cells from immunized mice by the method of Lopes and Alves (22). For the first fusion, hybridomas were distributed on 48-well plates (Costar), and at 10 days postfusion the colonies were screened by immunoblotting, as described below. For the second fusion, hybridomas were distributed on 96-well plates (Costar), and screening was performed by an enzyme immunoassay, as described else-where (18). After the hybridomas were cloned by limiting dilution and expansion of the positive clones, large amounts of antibodies were obtained from the ascitic fluid induced in BALB/c mice injected with Pristane (Sigma) before inoculation of hybridoma cells.
Antibody screening by immunoblotting.Exoantigens were submitted to SDS-PAGE with 10% acrylamide under reducing conditions, and proteins were then transferred to nitrocellulose membranes (Sigma). Fragments of nitrocellulose membranes containing gp70 were cut into small pieces and distributed in 48-well plates (Costar). After the free sites were blocked with PBS containing 5% skim milk, 200l of the supernatants from the hybridoma cultures was added to each well. After 1 h of incubation at room temperature, the wells were washed five times for 5 min each time with PBS containing 0.1% Tween 20 (Sigma) (PBS-T) and treated with affinity-purified peroxidase-conjugated goat anti-mouse immu-noglobulin (Bio-Rad) for 1 h at room temperature. The wells were again washed with PBS-T five times. The presence of reactive immunoglobulin G (IgG) was tested for by the addition of 4-chloro-naphthol in 50 mM Tris buffer (pH 6.8)– methanol–H2O2, and the IgG was finally washed with distilled water.
Pretreatment of immune sera for use in inh-ELISA.Aliquots of immune serum, CSF, or BAL fluid (200l) were mixed with an equal volume of 0.1 M EDTA (pH 7.2; Sigma) and boiled at 100°C for 3 to 5 min. The tubes were cooled and centrifuged at 13,000⫻gfor 30 min, and the supernatant was used for the test.
Inh-ELISA.The inh-ELISA was performed as described previously (19, 25). A standard inhibition curve was first prepared by adding known concentrations of gp70 to pooled normal human serum (NHS), CSF, or BAL fluid (a standard curve was prepared for each biological material) (25). The inhibition reaction occurred when constant aliquots of anti-gp70 MAb were mixed with the inhibi-tion standards, PCM patient serum, CSF and BAL fluid samples, and NHS control samples. These samples were then plated on previously blocked micro-titer plates (inhibition plates) and incubated overnight at 4°C. The reaction plates were coated with gp70, incubated overnight at 4°C, and blocked; and samples from each well in the inhibition plates (containing a mixture of MAb bound to circulating antigen and free MAb) were transferred to the respective wells in the reaction plates. The plates were washed and probed with goat anti-mouse IgG-peroxidase conjugate and developed with a chromogenic sub-strate, as described previously (19, 25). The optical density (OD) readings at 492 nm were then plotted on a standard curve constructed from the data derived from MAb titration with the inhibition standards. The antigen concentrations in serum, BAL fluid, and CSF samples were calculated by using a regression model constructed with the reciprocal values of the fixed concentrations of gp70 and the OD values. All of the standards, samples, and controls were tested in duplicate. The cutoff point was established as the receiver operator characteristic (ROC) curve.
the antigen concentrations and the OD values obtained. Comparisons were made by one-way analysis of variance.
RESULTS
MAb production.
The anti-gp70 MAb belongs to the IgG1
subclass and recognizes an antigenic determinant of
P.
brasil-iensis
with a relative molecular mass of 70 kDa. This MAb did
not recognize antigens from
Histoplasma capsulatum
or
Cryp-tococcus neoformans
.
Detection of
P. brasiliensis
gp70 in serum samples.
The
stan-dard curve was obtained by adding increasing concentrations
of gp70 to a pool of control NHS samples. The sensitivity
ranged from 0.001 to 30
g/ml. The cutoff point was
estab-lished from the ROC curve and was based on the antigen
concentration in the sera of patients with PCM and NHS.
Samples with antigen concentrations greater than 1.0
g/ml
were considered positive.
gp70 was detected in 80 of 81 patients (98.76%) with active
PCM (mean concentration, 8.19
g/ml) (Fig. 1). Among this
group, 11 patients presented with the acute form, 6 presented
with the chronic unifocal form, and 63 presented with the
chronic multifocal form; and the mean antigen concentrations
in these three groups were 11.86, 4.83, and 7.87
g/ml,
respec-tively (Fig. 2). Positive results were observed for all patients
with the acute or chronic unifocal form and 98.43% of the
patients with the chronic multifocal form (Table 1). No
cross-reactivity with sera from patients with histoplasmosis or
cryp-tococcosis or NHS was observed. Overall, the sensitivity and
specificity were 98.8 and 100%, respectively.
Detection of
P. brasiliensis
gp70 in CSF and sera from
pa-tients with neuro-PCM.
A standard inhibition curve was
con-structed with known concentrations of gp70 diluted in CSF
from patients with no fungal diseases. The cutoff value was
determined to be 0.21
g/ml for CSF. gp70 was detected in all
CSF samples, and the mean antigen concentration was 6.78
g/ml, showing 100% sensitivity and 100% specificity. Ten of
11 serum samples from patients with neuro-PCM had
detect-able antigen (mean concentration, 3.97
g/ml; cutoff value,
0.97
g/ml) (Table 2 and Fig. 3). No false-positive reactions
with control samples were observed.
Detection of
P. brasiliensis
gp70 in BAL fluid.
A standard
inhibition curve was constructed with known quantities of gp70
diluted in BAL fluid samples from patients with no fungal
diseases. The cutoff level for positivity was established to be
0.46
g/ml. All BAL fluid samples were positive for gp70, and
the mean antigen concentration for this group was 7.5
g/ml.
BAL fluid samples from individuals with no known infectious
diseases were negative for antigen detection (Table 2; Fig. 3).
gp70 antigen clearance during treatment with ITZ.
Table 3
shows the initial clinical manifestations of all 23 patients with
PCM as well as the gp70 antigen concentrations obtained for
serum samples collected at the baseline and at the end of
treatment. All 23 serum samples from patients with PCM had
levels of circulating gp70 antigen above the cutoff point at the
time of diagnosis, with a mean antigen concentration of 8.23
g/ml. Antigen levels in serum samples collected at the first
visit after the start of therapy (30 days after the start of
anti-fungal therapy) dropped to a mean concentration of 4.0
g/ml
and further decreased by the 12th month, when the mean
antigen concentration was 0.30
g/ml. Of interest, all but four
FIG. 1. Detection of circulating antigen in sera from patients with
PCM or other mycoses and in NHS by inh-ELISA. Groups studied: 1,
patients with PCM (
n
⫽
81); 2, patients with cryptococcosis (
n
⫽
20);
3, patients with histoplasmosis (
n
⫽
51); 4, NHS (
n
⫽
93). Bars
represent the mean antigen concentration for each group. The long
fine line represents the cutoff point (1.0
g/ml).
FIG. 2. Detection of circulating antigen in sera from patients with
different forms of PCM by inh-ELISA. Groups studied: 1, patients with
acute form of PCM (
n
⫽
11); 2, patients with chronic unifocal form of
PCM (
n
⫽
6); 3, patients with chronic multifocal form of PCM (
n
⫽
64). Bars represent the mean antigen concentration for each group.
The long fine line represents the cutoff point (1.0
g/ml).
TABLE 1. Detection of
P. brasiliensis
70-kDa circulating antigen in
sera from patients with PCM and other fungal infections and sera
from healthy controls by inh-ELISA
Serum group
No. of serum samples % Reactive
samples Mean antigen
concn (g/ml) Total Reactivea Nonreactive
PCM
Acute
11
11
0
100
11.86
Chronic
unifocal
6
6
0
100
4.83
Chronic
multifocal
64
63
1
98.43
7.87
Histoplasmosis
51
0
51
0
0
Cryptococcosis
20
0
20
0
0
NHS
93
0
93
0
0
aA sample was considered positive (i.e., reactive) if the antigen concentration
serum samples collected at the end of antifungal treatment
showed complete antigen clearance and exhibited negative
re-sults (Fig. 4).
Overall, the patients showed a significant reduction (
P
⬍
0.0001) in the levels of circulating gp70 by the third month
after the initiation of therapy, and this reduction was
main-tained until the end of the follow-up period (Fig. 5). All
pa-tients improved clinically and mycologically with therapy. The
antibody titers determined by the ID test at the time of
diag-nosis varied from 1:2 to 1:16, and in almost all patients the
titers decreased during the course of treatment.
DISCUSSION
Since the demonstration of the antigenemia produced in
animals experimentally infected with
Streptococcus
pneu-moniae
(9), there have been numerous reports on the detection
of antigens in blood, urine, and CSF of patients and animals
infected with bacteria, fungi, viruses, and parasites. In the case
of fungi, with the exception of the capsular polysaccharide of
C. neoformans
, which has been detected since 1950 (10),
ad-vances have required sensitive immunoassay procedures that
have become widely applied only recently. In
immunocompro-mised patients with cryptococcosis, histoplasmosis, or
aspergil-losis, the detection of circulating antigens can provide a rapid
diagnosis and prompt treatment in the first days of infection,
when antibodies are not yet produced or are present at low
levels (11, 12). Detection of circulating
H. capsulatum
antigen
has proved to be useful for the diagnosis and follow-up of
patients with histoplasmosis (42, 43).
Very few techniques have shown high sensitivities and
spec-ificities for antigen detection in biological specimens for the
diagnosis and follow-up of PCM patients. In our previous
stud-ies, we successfully detected gp43 in sera, CSF, and BAL fluid
samples from PCM patients by inh-ELISA, which proved to be
suitable for the monitoring of patients receiving antimycotic
therapy. Go
´mez et al. (19, 20) used the inh-ELISA to detect
the 87-kDa molecule in the sera of patients with PCM for both
diagnostic and follow-up purposes. Antibody detection systems
have also been used to assay other molecules for the diagnosis
of PCM, such as a 22- to 25-kDa protein (14), a 58-kDa
gly-coprotein (15), and an 87-kDa protein (8).
In the present study, the overall sensitivity of gp70 detection
among the 81 PCM patients was 98.8% in the presence of a
mean antigen concentration of 8.19
g/ml and reached 100%
in patients with the acute form of the disease, with a mean
antigen concentration of 11.86
g/ml. Among patients with the
chronic form of PCM, antigenemia due to gp70 was observed
in 98.43% of serum samples from patients with the multifocal
form (mean antigen level, 7.87
g/ml); on the other hand, the
sera of 100% of the patients with the chronic unifocal form had
gp70, with a mean antigen concentration of 4.83
g/ml. gp70
was detected in 100% of the CSF and BAL fluid samples, with
mean antigen concentrations of 6.78 and 7.5
g/ml,
respec-tively. No cross-reactions were observed when sera from
pa-tients with other mycoses were tested. The majority (90.9%) of
serum samples from patients with neuro-PCM were positive
for gp70, with a mean antigen concentration of 3.97
g/ml. The
inh-ELISA proved to be helpful in detecting
P. brasiliensis
gp70 in biological samples, including BAL fluid and CSF, with
100% sensitivity. Considering the limitations of the
conven-tional methods for the diagnosis of neuro-PCM, our findings
suggest that the gp70 antigen test may be useful for
confirma-tion of the diagnosis in such patients. In addiconfirma-tion, the
inh-ELISA has the advantage of being able to process large
num-bers of samples at the same time and presents a high sensitivity
and a high specificity.
For diagnostic purposes, tests based on the ID test are
gen-erally considered to have high specificities; however,
false-FIG. 3. Detection of gp70 antigen in CSF from patients with
neuro-PCM and BAL fluid from patients with pulmonary neuro-PCM by
inh-ELISA. Groups studied: 1, CSF of patients with PCM (
n
⫽
14); 2, CSF
of patients with no fungal disease (
n
⫽
6); 3, BAL fluid from patients
with PCM (
n
⫽
23); 4, BAL fluid from patients with no fungal
pul-monary disease (
n
⫽
10); 5, serum from patients with neuro-PCM (
n
⫽
11). Bars represent the average antigen concentration for each
group.
TABLE 2. Detection of
P. brasiliensis
gp70 antigen by inh-ELISA in various specimens from patients with PCM and their respective controls
Specimen group No. of serum samples % Reactive samples Mean antigen concn (g/ml) Total Reactive Nonreactive
BAL fluid (from patients with PCM)
a23
23
0
100
7.5
CSF (from patients with PCM)
b14
14
0
100
6.78
BAL fluid (control)
10
0
10
0
0
CSF (control)
6
0
6
0
0
Serum from patients with neuro-PCM
c11
10
1
90.9
3.97
aA sample was considered positive (i.e., reactive) if the antigen concentration was
⬎0.46g/ml.
bA sample was considered positive (i.e., reactive) if the antigen concentration was
⬎0.21g/ml.
cA sample was considered positive (i.e., reactive) if the antigen concentration was
negative results may also occur, and this may be related to the
production of low-avidity IgG2 antibodies directed against
car-bohydrate epitopes (31). The sensitivities of ID test-based tests
range from 65 to 100% (1, 6, 29), depending on the antigen
preparation used. On the other hand, tests with higher
sensi-tivities, such as immunoenzymatic assays, present problems
associated with specificity due to cross-reactivity with
heterol-ogous sera (5, 19).
For many years, assessment of the clearance of the fungal
burden during treatment for PCM has been based on the
detection of antibodies against
P. brasiliensis
crude or purified
antigens (5, 29, 30, 39). Although healing of apparent fungal
lesions may occur within a short time after antifungal
treat-ment is initiated, long therapeutic courses are desirable in
order to prevent relapses. In this case, the status of the fungal
infection and the host response are usually monitored by
indi-rect methods, in which serology provides information about
the prognosis (5).
Our experience with the follow-up of patients receiving
treatment for PCM has shown that many times the antibody
titers obtained by the ID test do not correlate with the clinical
status of the patient. For example, in some patients elevated
antibody titers (1:64) were observed until the end of treatment,
when the patients were clinically cured. On the other hand,
although for most patients low antibody titers are related to
the absence of clinical symptoms (1:2 or 1:4), in some cases low
titers are present for months and are present in concert with
clinical symptoms. Such discrepancies between the clinical
sta-tus of the host and specific antibody detection are probably
related to the fact that the cell response and not the humoral
response is the main immunologic mechanism able to contain
P. brasiliensis
in the infected organism. For these reasons, the
use of serology as a single criterion of cure in patients with
PCM is controversial. In clinical practice, clinical, radiological,
mycological, and serological aspects must be evaluated over a
long period of observation in order to assess the results of
treatment.
Regarding the detection of antigen during treatment,
Mendes-Giannini et al. (28), working with a pool of sera from
patients with PCM using a Western blot assay, observed that
FIG. 4. Mean circulating antigen concentration in sera from
pa-tients with PCM at the time of diagnosis and during the period of ITZ
treatment. Error bars, standard deviation; m, months; n, number of
patients.
TABLE 3. Clinical manifestations and serology results for 23 patients evaluated before and after antifungal treatment
Patient no. and lesion
sitea No. of samples tested
Antigen level (g/ml) at: Antibody titer atb:
Diagnosis First follow-up End of treatmentc Diagnosis End of treatment
1. L, M, LN
8
6.75
5.01
0 (11)
1:8
NDS
2. L, M, LN
7
9.75
15.0
0 (9)
1:4
NR
3. L, M, LN
7
5.64
3.24
0 (9)
1:2
NR
4. L, ORP, LN
9
14.25
9.75
2.16 (12)
1:16
1:4
5. L, S, M, LN
7
13.5
3.39
0 (11)
1:16
1:2
6. L, M, LN
7
9.0
3.59
0 (9)
1:16
1:4
7. L, LN, Lx
6
11.25
1.77
0 (8)
1:4
NDS
8. L, M, LN
7
10.5
3.8
1.69 (9)
1:16
1:4
9. L, M, LN
7
4.16
7.26
0 (11)
NR
NR
10. L, M
7
7.5
2.67
0 (9)
1:16
1:4
11. L, M, LN
7
9.75
2.74
0 (10)
1:8
1:2
12. L, M, LN
6
4.53
2.37
0 (9)
1:2
NR
13. L, M
6
7.12
3.20
1.03 (9)
1:4
1:2
14. L, M
5
6.94
3.69
0 (9)
1:2
NDS
15. L, M
6
3.57
1.76
0 (8)
1:4
NDS
16. L, M, LN, Lx
6
13.5
2.86
2.24 (9)
1:16
1:4
17. L, M
8
4.94
2.76
0 (10)
1:2
NDS
18. L, M
7
3.57
1.57
0 (10)
1:4
NDS
19. L, Lx
7
8.25
4.18
0 (10)
1:16
1:2
20. L, M
8
10.5
4.09
0 (10)
1:16
1:16
21. L, SNC
6
12.75
3.69
0 (12)
1:16
1:2
22. L, M
8
4.16
1.60
0 (12)
1:4
1:2
23. L, M, LN
7
7.5
2.13
0 (12)
1:4
NDS
Mean
6.91
8.23
4.0
0.30
aL, lung; M, mucosae; LN, lymph nodes; Lx, Larynx; ORP, oropharynx; S, skin; CNS, central nervous system. bNR, nonreactor; NDS, reactive only with nondiluted serum.
gp43 started to disappear from the circulation after 10 months
of treatment and was undetectable after 2 years of treatment.
More recently, the same group of investigators (36) detected
P.
brasiliensis
antigens in 75% of patient urine samples tested by
an indirect competition enzyme immunoassay and by an
im-munoblot test for monitoring the response to therapy.
One of the main goals of the present study was to assess
gp70 antigen clearance during treatment of PCM with ITZ by
comparing the clearance from serum samples collected at the
baseline and after 8 to 12 months of therapy. All 23 patients
selected for the study showed a good clinical response to
ther-apy, with no relapse of fungal disease documented over the
follow-up period. Overall, decreasing levels of
P. brasiliensis
gp70 were detected in patients during successful antifungal
therapy. In contrast, the ID test titers of these patients varied
over the follow-up period (1:16 to 1:2). Table 3 shows the
clinical manifestations of PCM in the patients and the
corre-lation between the levels of gp70 antigenemia and the levels of
anti-
P. brasiliensis
antibody at the time of diagnosis, 1 month
after treatment, and the end of treatment. At the time of
diagnosis, all except one of the patients had detectable gp70
antigen and anti
-P. brasiliensis
antibodies (one patient had a
negative ID test result). A significant decrease in gp70 titers
occurred during the first month of therapy in 22 patients; at the
end of therapy, only 4 (17.39%) patients (patients 4, 8, 13, and
16) had detectable gp70 antigen (antigen levels,
⬍
2.5
g/ml)
and positive titers by the ID test.
By the end of the follow-up period, antibody titers were
negative in three patients (patients 2, 3, and 12), and one
patient (patient 9) had undetectable antibodies (by the ID test)
from the time of diagnosis to the end of treatment. However,
the antigen level was also negative until the end of follow-up.
Once the levels of gp70 decreased, gp70 persisted at low levels
in serum until the end of therapy. On the other hand, in
various patients, the antibody titers seemed to decrease a little
later, after about 6 months of therapy. Of particular note is the
common persistence of antibodies for a considerable period of
time even after the cessation of an apparently successful course
of treatment.
Decreased levels of antigenemia were observed in two
pa-tients after 6 months of ITZ treatment, but their anti-
P.
bra-siliensis
antibody titers remained high (1:16) and did not
change throughout the period of treatment (patient 20). On
the other hand, patient 9 had undetectable antibodies from the
time of diagnosis to month 11 of treatment but had a
substan-tial decrease in antigenemia levels during treatment with ITZ.
All PCM patients showed clinical improvement after the
pe-riod of antifungal therapy (8 to 12 months).
In conclusion, our results indicate that the detection and
quantification of
P. brasiliensis
gp70 antigen in serum by
inh-ELISA is a sensitive method for the diagnosis of PCM and may
be used to evaluate patients for the clearance of the fungal
burden during treatment. Moreover, the high sensitivity of this
assay for the detection of gp70 in different biological fluids
opens a new possibility for the diagnosis of PCM in patients for
whom conventional methods present limitations.
ACKNOWLEDGMENTS
This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do
Estado de Sa˜o Paulo (FAPESP).
We thank Elettra Grene for correction of the English.
REFERENCES
1. Alves, J. R.1996. Comparac¸a˜o entre treˆs me´todos sorolo´gicos no seguimento de pacientes com paracoccidioidomicose. Master’s thesis. Universidade Fed-eral de Sa˜o Paulo, Sa˜o Paulo, Brazil.
2. Benard, G., M. J. S. Mendes-Giannini, M. Juvenale, E. T. Miranda, and A. J. S. Duarte.1997. Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to twoParacoccidioides brasiliensis glycoproteins that elicit strong humoral immune response. J. Infect. Dis.175:1263–1267. 3. Blotta, M. H. S. L., and Z. P. Camargo.1993. Immunological response to
cell-free antigens ofParacoccidioides brasilienis: relationship with clinical forms of paracoccidioidomycosis. J. Clin. Microbiol.31:671–676. 4. Bradford, M.1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye bind-ing. Anal. Biochem.72:248–254.
5. Brummer, E., E. Castan˜eda, and A. Restrepo.1993. Paracoccidioidomycosis: an update. Clin. Microbiol. Rev.6:89–117.
6. Camargo, Z. P., C. S. Unterkircher, and L. R. Travassos.1989. Identification of antigenic polypeptides ofParacoccidioides brasiliensisin serological tests. J. Med. Vet. Mycol.27:407–412.
7. Camargo, Z. P., R. Berzaghi, C. C. Amaral, and S. H. Marques da Silva. 2003. Simplified method for producingParacoccidioides brasiliensis exoanti-gens for use in immunodiffusion tests. Med. Mycol.41:539–542.
8. Díez, S., B. L. Go´mez, A. Restrepo, R. J. Hay, and A. Hamilton. 2002.
Paracoccidioides brasiliensis87 kilodalton antigen, a heat shock protein use-ful in diagnosis: characterization, purification and detection via immunohis-tochemistry. J. Clin. Microbiol.40:359–365.
9. Dochez, A. R., and O. T. Avery.1917. The elaboration of specific soluble substances byPneumococcusduring growth. J. Exp. Med.26:477–479. 10. Drouhet, E.1989. Overview of fungal antigens, p. 3–38.InE. Drouhet, G. T.
Cole, L. Repentigny, J. P. Latge´, and B. Dupont (ed.), Fungal antigen: isolation, purification, and detection. Plenum Publishing Corporation, New York, N.Y.
11. Drutz, D. J.1986. Antigen detection in fungal infections. N. Engl. J. Med. 314:115–117.
12. Ferreira-da-Cruz, M. F., B. Galva˜o-Castro, and C. T. Daniel-Ribeiro.1992. Isolation and antigenicity of a 45-kilodaltonParacoccidioides brasiliensis im-munodominant antigen. Infect. Immun.60:2667–2671.
13. Ferreira-da-Cruz, M. F., B. Galva˜o-Castro, and C. T. Daniel-Ribeiro.1991. Sensitive immunoradiometric assay for the detection ofParacoccidioides brasiliensisantigens in human sera. J. Clin. Microbiol.29:1202–1205. 14. Figueroa, J., A. J. Hamilton, M. H. Allen, and J. Hay.1994.
Immunohisto-chemical detection of a novel 22- to 25-kilodalton glycoprotein of Paracoc-cidioides brasiliensisin biopsy material and partial characterization by using species-specific monoclonal antibodies. J. Clin. Microbiol.32:1566–1574. 15. Figueroa, J., A. J. Hamilton, M. H. Allen, and J. Hay.1995. Isolation and
partial characterization of aParacoccidioides brasiliensis58 kDa extracellular glycoprotein which is recognized by human immune sera. Trans. R. Soc. Trop. Med. Hyg.89:566–572.
16. Freitas-da-Silva, G., and M. C. Roque-Barreira.1992. Antigenemia in para-coccidioidomycosis. J. Clin. Microbiol.30:381–385.
17. Garcia, N. M., G. B. Del Negro, H. P. Martins, and C. S. Lacaz.1987. Detection ofParacoccidioides brasiliensiscirculating antigens by immuno-electrophoresis-immunodiffusion technique. Preliminary report. Rev. Med. Trop. Sa˜o Paulo29:327–328.
18. Gesztesi, J.-L., R. Puccia, L. R. Travassos, A. P. Vicentini, J. Z. Moraes, M. F. Franco, and J. D. Lopes.1996. Monoclonal antibodies against the 43,000 kDa glycoprotein fromParacoccidioides brasiliensismodulate lami-nin-mediated fungal adhesion to epithelial cells and pathogenesis. Hybrid-oma15:415–422.
19. Go´mez, B. L., J. L. Figueroa, A. J. Hamilton, B. Ortiz, M. A. Robledo, R. J. Hay, and A. Restrepo.1997. Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens. J. Clin. Microbiol.35:3278–3283.
20. Go´mez, B. L., J. I. Figueroa, A. J. Hamilton, S. Diez, M. Rojas, A. M. Tobo´n, R. J. Hay, and A. Restrepo.1998. Antigenemia in patients with paracoccid-ioidomycosis: detection of the 87-kilodalton determinant during and after antifungal therapy. J. Clin. Microbiol.36:3309–3316.
21. Kurokawa, C. S., M. F. Sugisaki, and M. T. S. Perac¸oli.1998. Virulence factors in fungi of systemic mycosis. Rev. Inst. Med Trop. Sa˜o Paulo40:125– 135.
22. Lopes, J. D., and, M. G. M. Alves.1983. Production of monoclonal antibod-ies by somatic cells hibridization, p. 386–398.InC. M. Morel (ed.), Genes and parasites: a laboratory manual. Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil.
23. Magaldi, S. W., and D. W. R. Mackenzie.1986. Deteccio´n de antigenemia y anticuerpos deParacoccidioidesmediante procedimentos electrofore´ticos invertidos, p. 80.InProceedings Coloquio Internacional sobre la Paracoc-cidioidiomicosis. Corporacio´n para Investigaciones Biolo´gicas, Medellín, Colombia.
24. Magaldi, S. W., D. W. R. Mackenzie, and M. B. Albornoz.1989. Detection of
Paracoccidioides brasiliensiscirculating antigen by the passive hemagglutina-tion inhibihemagglutina-tion in patients sera, p. 18.InProceedings Encuentro Interna-tional sobre Paracoccidioidiomicosis. Instituto Venezolano de Investigacio-nes Cientificas, Caracas, Venezuela.
25. Marques da Silva, S. H., A. L. Colombo, M. H. S. L. Blotta, L. D. Lopes, F. Queiroz-Telles, and Z. P. de Camargo.2003. Detection of circulating gp43 antigen in serum, cerebrospinal fluid and bronchoalveolar lavage of patients with paracoccidioidomycosis. J. Clin. Microbiol.41:3675–3680.
26. Marques da Silva, S. H., F. Queiroz-Telles, A. L. Colombo, M. H. S. L. Botta, J. D. Lopes, and Z. P. Camargo.2004. Monitoring gp43 antigenemia in
paracoccidioidomycosis patients during therapy. J. Clin. Microbiol.42:2419– 2424.
27. Mattos Grosso, D., S. R. de Almeida, M. Mariano, and J. D. Lopes.2003. Characterization of gp70 and anti-gp70 monoclonal antibodies in Paracoc-cidioides brasiliensispathogenesis. Infect. Immun.71:6534–6542.
28. Mendes-Giannini, M. J. S., J. P. Bueno, M. A. Shikanai-Yasuda, A. W. Ferreira, and A. Masuda.1989. Detection of the antigen 43,000-molecular-weight glycoprotein in sera of patients with paracoccidioidomycosis. J. Clin. Microbiol.27:2842–2845.
29. Mendes-Giannini, M. J. S., J. P. Bueno, M. A. Shikanai-Yasuda, A. M. S. Stolf, A. Masuda, V. Amato-Neto, and A. W. Ferreira.1990. Antibody re-sponse to the 43kDa glycoprotein ofParacoccidioides brasiliensisas a marker for the evaluation of patients under treatment. Am. J. Trop. Med. Hyg. 43:200–206.
30. Mendes-Giannini, M. J. S., G. B. Del Negro, and A. M. Siqueira.1994. Serodiagnosis, p. 345–363.InM. Franco, C. S. Lacaz, A. Restrepo, and G. Del Negro (ed.), Paracoccidioidomycosis. CRC Press, Inc., Boca Raton, Fla. 31. Neves, A. R., R. L. Mamoni, C. L. Rossi, Z. P. Camargo, and M. H. S. L. Blotta.2003. Negative immunodiffusion test results obtained with sera of paracoccidioidomycosis patients may be related to low-avidity immunoglob-ulin G2 antibodies directed against carbohydrate epitopes. Clin. Diagn. Lab. Immunol.10:802–807.
32. Puccia, R., and L. R. Travassos.1991. 43-Kilodalton glycoprotein from
Paracoccidioides brasiliensis: immunochemical reactions with sera from pa-tients with paracoccidioidomycosis, histoplasmosis, or Jorge Lobo’s disease. J. Clin. Microbiol.29:1610–1615.
33. Puccia, R., S. Schenkman, P. A. Gorin, and L. R. Travassos.1986. Exocel-lular components ofParacoccidioides brasiliensis: identification of a specific antigen. Infect. Immun.53:199–206.
34. Restrepo, A.2000.Paracoccidioides brasiliensis, p. 2768–2772.InG. L. Man-dell, J. E. Bennett, and R. Rolin (ed.), Principles and practice of infectious disease, 5th ed. Churchill Livingstone, Philadelphia, Pa.
35. Rodrigues, M. C., C. M. Cassaguerra, and C. S. Lacaz.1984. Antigenemia in paracoccidioidomycosis. Probable demonstration of circulating antigen by counterimmunoelectrophoresis test. Rev. Inst. Med. Trop. Sa˜o Paulo26: 285–287.
36. Salina, M. A., M. A. Shikanai-Yasuda, R. P. Mendes, B. Barravieira, and M. J. S. Mendes-Giannini.1998. Detection of circulatingParacoccidioides brasiliensisantigen in urine of paracoccidioidomycosis patients before and during treatment. J. Clin. Microbiol.36:1723–1728.
37. San-Blas, G., F. San-Blas, and L. E. Serrano.1977. Host-parasite relation-ship in the yeast-like form ofParacoccidioides brasiliensisstrain IVIC Pb9. Infect. Immun.15:343–346.
38. San-Blas, G., and F. San-Blas.1982. Variability of cell wall composition in
Paracoccidioides brasiliensis: a study of two strains. Sabouraudia20:31–40. 39. Siqueira, A. M.1982. Diagno´stico imunolo´gico, p. 253–264.InG. Del Negro,
C. S. Lacaz, and A. M. Forillo (ed.), Paracoccidioidomicose. Sarvier-Editora da Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil.
40. Stambuk, B. U., R. Puccia, M. L. C. Almeida, L. R. Travassos, and S. Schenkman.1988. Secretion of the 43 kDa glycoprotein antigen by Paracoc-cidioides brasiliensis.J. Med. Vet. Mycol.26:367–373.
41. Svidzinski, T. I. E., M. H. Miranda-Neto, R. G. Santana, O. Fischman, and A. L. Colombo.1999.Paracoccidioides brasiliensisisolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage. Rev. Inst. Med. Trop. Sa˜o Paulo41:279–283. 42. Wheat, L. J., R. B. Kohler, and R. P. Tewari.1986. Diagnosis of disseminated
histoplasmosis by detection ofHistoplasma capsulatumantigen in serum and urine specimens. N. Engl. J. Med.314:83–88.
43. Wheat, L. J., H. Wheat, P. Connoly, M. Kleiman, K. Supparatpinyo, K. Nelson, R. Bradsher, and A. Restrepo.1997. Cross-antigen inHistoplasma capsulatumvarietycapsulatumantigen assays of urine samples from patients with endemic mycoses. Clin. Infect. Dis.24:1169–1171.
