1556-6811/09/$12.00 doi:10.1128/CVI.00176-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
New Approach for Diagnosis of Candidemia Based on
Detection of a 65-Kilodalton Antigen
䌤
Rodrigo Berzaghi,
1Arnaldo Lopes Colombo,
2Antonia Maria de Oliveira Machado,
3and Zoilo Pires de Camargo
1*
Department of Microbiology, Immunology and Parasitology,
1Department of Medicine, Infectious Diseases Section,
2and
Department of Medicine, Central Laboratory, Microbiology Division,
3Federal University of Sa
˜o Paulo, Sa
˜o Paulo, Brazil
Received 27 April 2009/Returned for modification 27 May 2009/Accepted 16 September 2009
Nosocomial candidiasis is a major concern in tertiary care hospitals worldwide. This infection generally
occurs in patients with degenerative and neoplastic diseases and is considered the fourth most frequent cause
of bloodstream infections. Diagnosis of candidemia or hematogenous candidiasis has been problematic
be-cause clinical signs and symptoms are nonspecific, leading to delays in diagnosis and, consequently, delays in
appropriate antifungal therapy. We developed an inhibition enzyme-linked immunosorbent assay (ELISA) for
detection of a 65-kDa antigen in an experimental model of candidemia and for diagnosis of patients in intensive
care units (ICUs) with suspected candidemia. An anti-65-kDa monoclonal antibody was tested for detection of
the 65-kDa antigen produced by
Candida albicans
,
Candida tropicalis
, and
Candida parapsilosis
in murine
candidemia models. The 65-kDa antigen was detected in sera at concentrations ranging from 0.012 to 3.25
g/ml. A total of 20 human patients with candidemia were then evaluated with the inhibition ELISA using
sequential sera. Sixteen (80%) patients had the 65-kDa antigen in concentrations ranging from 0.07 to 5.0
g/ml. Sequential sera from patients with candidemia presented three different patterns of antigenemia of the
65-kDa molecule: (i) total clearance of antigenemia, (ii) initial clearance and relapse of antigenemia, and (iii)
partial clearance of antigenemia. Our results indicate detection of the 65-kDa protein may be a valuable tool
for the diagnosis of candidemia by
C. albicans
,
C. tropicalis
, and
C. parapsilosis
.
Nosocomial candidiasis is a major concern in tertiary care
hospitals worldwide. This infection generally occurs in patients
with degenerative and neoplastic diseases exposed to
broad-spectrum antibiotics, immunosuppressive drugs, and invasive
medical procedures (10, 11, 32, 40, 48). The incidence of
in-vasive candidiasis has increased over the past two decades, and
candidemia is now considered the fourth most frequent cause
of bloodstream infections (50, 53). A recent nationwide
sur-veillance study, conducted in public general tertiary care
hos-pitals in Brazil, found an incidence of 2.69 episodes/1,000
ad-missions, a rate 2 to 8 times higher than that observed in
medical centers from Northern Hemisphere countries (10). In
South America, most candidemic episodes are related to
Can-dida albicans
,
Candida tropicalis
, and
Candida parapsilosis
strains;
Candida glabrata
has been scarcely reported (9–12, 15,
21, 25, 42, 55). This is in contrast to United States and
Euro-pean medical centers, where
C. glabrata
is considered a major
pathogen.
Diagnosis of candidemia or hematogenous candidiasis has
been problematic. The clinical signs and symptoms are
non-specific; therefore, the diagnosis and, consequently,
appropri-ate antifungal therapy are delayed. Even in patients with
au-topsy-proven systemic candidiasis, positive diagnoses from
blood cultures ranged from 40 to 60% (51–53).
Antigen detection for the serodiagnosis of invasive
Candida
infections has been reported (5, 6, 13, 14, 18–20). Matthews
and Burnie developed an immunobinding method for
detec-tion of a 47-kDa cytoplasmatic protein antigen in patients with
systemic candidiasis (34). An immunoassay detecting a 48-kDa
antigen of
Candida
, subsequently recognized as enolase (13,
33), is available for the diagnosis of invasive candidiasis (54).
Latex agglutination tests are based on the detection of
man-nan, a cell wall component that is the most widely studied
antigen in patients with candidiasis (5, 20). Commercial tests
(Pastorex
Candida
assay and Cand-Tec assay) have been used
to detect this molecule in sera. Colorimetric assays (Fungitec
G and Fungitec G MT) detect

-
D-glucan, a major structural
component of the fungal cell wall, in serum and have been used
for diagnosis of fungal infections. Studies show the
concentra-tion of

-
D-glucan is increased in experimental models of
fun-gal infections (35–38), as well as in the plasma of patients with
mycosis (20, 36).
Various tests have been developed based on detection of
antibodies, antigens, and metabolites, although, they are all
time-consuming and lack either specificity or sensitivity (50). In
C. albicans
, a 65-kDa mannoprotein (Mp65) is a structural and
secreted component of the fungus. This mannoprotein is
par-ticularly observed in extracellular fractions of hyphal cells (1, 8,
17, 44–46). Mp65 is also present in both the structural and
secretory mannoprotein material and is recognized by
periph-eral blood T cells of practically all healthy individuals
(quasi-universal antigen) (1, 44, 45), making it a potential target for
immunodiagnosis of patients with suspected candidemia.
Arancia et al. (1) used real-time PCR to detect and quantify
C.
albicans
in sera from patients with invasive candidiasis. This
assay was specific for a DNA fragment containing the gene for
the 65-kDa mannoprotein of
C. albicans
(Ca
MP65
). The assay
* Corresponding author. Mailing address: Department of
Microbi-ology, Immunology and ParasitMicrobi-ology, Federal University of Sa˜o Paulo,
Sa˜o Paulo, Brazil. Phone: 11 55 11 5576 4523. Fax: 11 55 11 5571 5877.
E-mail: zpcamargo@unifesp.br.
䌤
Published ahead of print on 23 September 2009.
was shown to be sensitive and specific for
C. albicans
, allowing
quantitative detection of this fungus in clinical samples.
The inhibition enzyme-linked immunosorbent assay
(inh-ELISA) is an enzyme immunoassay (EIA) to detect circulating
antigens in sera of patients with invasive fungal infections. This
test uses a species-specific murine monoclonal antibody (MAb)
with high sensitivity and specificity and is useful in diagnosis
and follow-up of paracoccidioidomycosis patients. It is also
useful for antigen detection in the cerebrospinal (28) and
bron-choalveolar (27, 29–31) fluids of these patients.
In this study, our intention was to demonstrate that
inh-ELISA is a sensitive detection method able to detect
nano-grams of antigen in serum samples from patients with
candi-demia and not to compare it or show its superiority to other
assays. Na and Song previously compared three serological
methods of detecting
C. albicans
secreted aspartyl proteinase
antigen (39); they found inh-ELISA had 93.9% sensitivity and
96.0% specificity and detected concentrations ranging from 6.3
to 19.0 ng/ml. The sensitivity and specificity for standard
ELISA were 69.7 and 76.0%, respectively; while for capture
ELISA, the sensitivity and specificity were 93.9 and 92.0%,
respectively. The results of Na and Song showed inh-ELISA
with MAb CAP1 effectively detected circulating secreted
as-partyl proteinase antigen and suggest it may be useful for the
diagnosis and treatment monitoring of invasive candidiasis.
The aim of this study was to standardize an alternative
inh-ELISA for detection of a 65-kDa antigen, present in
C.
albi-cans
,
C. tropicalis
, and
C. parapsilosis
, using a MAb specific for
the 65-kDa
Candida
protein. The assay could be used for
diagnosis and follow-up of patients with candidemia. The
present study involved five different stages: (i) identification of
an immunodominant 65-kDa antigen of
C. albicans
that is
common to
C. tropicalis
and
C. parapsilosis
, (ii) production
of an anti-
C. albicans
65-kDa-molecule MAb for detection
of the immunodominant antigen mentioned above, (iii)
ap-plication of the MAb to the inh-ELISA, (iv)
characteriza-tion of antigenemia in an animal model, and (v) evaluacharacteriza-tion
of the developed inh-ELISA with sera from patients with
candidemia.
MATERIALS AND METHODS
Fungal isolates.Isolates ofC. albicans(ATCC 90028),Candida parapsilosis (ATCC 22019),Candida tropicalis(ATCC 750), andCandida glabrata(ATCC 90030) were obtained from the yeast stock collection of the Special Mycology Laboratory, Federal University of Sa˜o Paulo.
Candidaexoantigens.EachCandidaspecies was grown on Sabouraud agar (three tubes) for 3 days at 36°C. All growth was transferred to a 250-ml Erlen-meyer flask containing 50 ml modified Lee’s medium without amino acids (MLMwAA) (23) under agitation (50 rpm). MLMwAA, as modified by Tronchin et al. (49), contains 5.0 g/liter (NH4)2SO4, 0.2 g/liter MgSO4䡠7H2O, 2.5 g/liter
K2HPO4, 5.0 g/liter NaCl, 10.0 g/liter glucose, and 0.04 g/liter biotin at pH 6.8.
This constituted a preinoculum, which was then transferred to a 1-liter Erlen-meyer flask containing the above medium for 7 days at 36°C under agitation (50 rpm). Then, the growth was killed with merthiolate (0.2 g/liter) and filtered. The filtrate was concentrated under vacuum at 45°C to a volume of 30 ml and dialyzed against distilled water for 48 h. Protein content was determined by the method of Bradford (4).
Exoantigens of heterologous fungi.Exoantigens ofHistoplasma capsulatum, Aspergillus fumigatus, andParacoccidioides brasiliensiswere prepared according to Smith and Goodman (43), Biguet et al. (3), and Camargo et al. (7), respec-tively.Sporothrix schenckiiandTrichosporum rubrumexoantigens were obtained according to the protocol described by Camargo et al. for Paracoccidioides brasiliensis(7). Protein content was determined by the method of Bradford (4).
SDS-PAGE and Western blot for determination of immunodominant anti-gens.The exoantigens obtained from theC. albicans,C. tropicalis,and C. parap-silosiscultures underwent sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) (22) and Western blotting as previously described (47). Briefly, after transferring proteins to nitrocellulose membrane, the membrane was blocked with Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.2) containing 5% nonfat dry milk for 2 h at room temperature. The membranes were then incubated with previously obtained rabbit anti-C. albicans hyperim-mune sera (1:50 dilution) for 1 h at room temperature. Next, the membranes were washed three times with phosphate-buffered saline (PBS)–Tween 20 and incubated with a 1:1000 dilution of peroxidase-conjugated anti-rabbit immuno-globulin G (IgG) (Sigma-Aldrich) for 1 h at 37°C. The reaction was developed in a solution containing 5 mg of 3,3⬘-diaminobenzidine (DAB; Sigma) and 10l of H2O2in 50 ml of Tris buffer, pH 7.4. The membranes were washed, dried,
analyzed, and stored for documentation.
Electroelution ofC. albicans65-kDa protein.Various SDS-PAGE gels ofC. albicansexoantigens were run and stained with 0.3 M copper chloride (CuCl2).
The region containing the 65-kDa molecule was excised from gels and electro-eluted in an electroelution chamber (Isco model 1750 electrophoretic concen-trator; Isco, Inc., Lincoln, NE). Electroelution was performed for 3 to 5 h in 192 mM glycine and 25 mM Tris-HCl, pH 8.3, at a 2-A constant current. Proteins were eluted and dialyzed exhaustively against distilled water. The concentration was determined by the method of Bradford (4).
MAb production of the anti-65 kDa C. albicansmolecule.The MAb was produced by the method of Lopes and Alves (26). Of note, the production of MAb was only initiated after a pilot study showed the antigen was recognized by polyclonal antibody. Six-week-old BALB/c mice were immunized every week for 3 weeks subcutaneously with 50g ofC. albicans65-kDa protein in PBS incor-porated into Freund’s complete adjuvant for the first injection and in incomplete Freund’s adjuvant for the subsequent ones. Injections were always made at four different sites in the axillary and inguinal regions at final volumes of 100l per site. Before each immunization, mice were bled through the ocular plexus and the serum was separated by centrifugation and stored at⫺20°C. Final immuni-zations (50g of 65-kDa protein in 100l of PBS intravenously) were performed 2 days before cell fusion, according to the method of Lopes and Alves (26). Positive colonies were screened by an EIA, as described below. After cloning by limiting dilution and expanding positive clones, large amounts of antibodies were obtained by producing ascites in BALB/c mice previously primed with Pristane (Sigma). MAbs were purified from culture supernatants and ascites by affinity chromatography on a protein A column. Ig isotyping was performed with the Clone Selector mouse MAb screening kit (Bio-Rad) according to the manufac-turer’s instructions. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Federal University of Sa˜o Paulo.
Antibody screening by EIA.The EIA was performed as described by Puccia and Travassos (41). Briefly, polyvinyl microplates (Corning Costar) were coated with 50l of a 2-g/ml solution of purified 65-kDa protein in PBS for 1 h at room temperature. After blocking free sites with PBS containing 5% nonfat milk for 2 h at room temperature, 50l of culture supernatant or purified MAb was added to each well. After 1 h of incubation at 37°C, wells were thoroughly washed with PBS containing 0.5% gelatin (Difco) and 0.05% Tween 20 (Sigma) (PBS-T-G) and treated with affinity-purified peroxidase-conjugated goat anti-mouse Ig (Bio-Rad) for 1 h at 37°C. This was followed by three washes with PBS-T-G. Reactions were developed by the addition ofo-phenylenediamine in 0.1 M acetate-phosphate buffer, pH 5.8, stopped with 4 N sulfuric acid, and read in a Titertek Multiskan EIA reader at 492 nm.
Specificity of MAb against 65-kDa protein.The exoantigens fromC albicans, C. tropicalis, C. parapsilosis,H. capsulatum, A. fumigatus, P. brasiliensis,S. schenckii, andT. rubrumwere subjected to SDS-PAGE (22) and Western blot-ting (47). Briefly, the membrane was blocked with Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.2) containing 5% nonfat dry milk for 2 h at room temperature. The membrane was then incubated with anti-65 kDa MAb (10
g/ml) for 1 h at room temperature. Then, the membrane was washed three times with PBS-Tween 20 and incubated with a 1:1,000 dilution of peroxidase-conjugated anti-mouse IgG (Sigma) for 1 h at 37°C. The reaction was developed in a solution containing 5 mg of DAB (Sigma) plus 10l of H2O2in 50 ml of Tris
buffer, pH 7.4. The membrane was washed, dried, and stored for documentation.
inh-ELISA for 65-kDa molecule detection.For detection of the 65-kDa mol-ecule by inh-ELISA, we followed a method described by Go´mez et al. (16) and Marques-da-Silva et al. (33), based on the method described by Le Pape and Deunff (24). An inh-ELISA was developed for serum samples. The diluting buffer used in the serum experiments consisted of a pool of normal human serum (NHS; 1:10 dilution) in 0.05% PBS–Tween 20 (PBS-Tween) and 20 mM MgCl2.
Purified anti-65 kDa MAb was used (10 mg/ml), and all samples were diluted 1:2 in diluting buffer. The method is illustrated in detail in Fig. 1.
Pretreatment of immune sera for use in inh-ELISA.Aliquots of immune serum (200l) were mixed with an equal volume of 0.1 M EDTA (Sigma), pH 7.2, and boiled at 100°C for 3 to 5 min. The tubes were cooled and centrifuged at 13,000⫻gfor 30 min. The supernatant was used for the test.
Inhibition plates.An inhibition standard curve was constructed by adding different concentrations ofC. albicans65-kDa protein (from 1 ng/ml to 30g/ml) to 100l of pooled NHS and then adding 100l of the standardized concen-tration of anti-65 kDa protein MAb. NHS (diluted 1:2 in diluting buffer) was used as a negative control. All standards, samples, and controls were tested in triplicate. Samples were plated onto 96-well flat microtiter plates (Corning Costar), previously blocked with 200l of 5% nonfat milk per well, made up in PBS-Tween, for 2 h at 37°C. Plates were mixed in a shaker for 30 min at room temperature and then incubated overnight at 4°C.
Reaction plates.Maxisorp polystyrene plates (Corning Costar) were coated with 500 ng ofCandida albicans65-kDa protein in 0.06 M carbonate buffer, pH 9.6 (100l/well). The plates were left at room temperature for 30 min and then incubated overnight at 4°C. After incubation, the plates were washed three times with PBS-Tween and blocked with 200l per well of 1% bovine serum albumin in PBS for 1 h at 37°C. After three more washes, 100l from each inhibition plate well (containing a mixture of the MAb circulating complexes and free MAb) was transferred to the respective wells in the reaction plate and incubated for 2 h at 37°C. After being washed as described above, 100l of goat anti-mouse IgG-peroxidase (Sigma) was added, and the plates were incubated for 1 h at 37°C. After further washings, the reaction was developed with SuperSignal West Pico Chemiluminescent solution (1:10) (Pierce biotechnology). Optical densities (ODs) were measured at 470 nm with a Chemiluminescent ELISA reader (Spec-traMax). The OD at 470 nm was then plotted on a standard curve constructed from the data derived from MAb titration with NHS containing known quantities of 65-kDa protein, as described above. The degree of inhibition of MAb binding was shown to be reciprocal to the concentration of circulating antigen in the sample. The cutoff point was established as the receiver operator characteristic curve.
Neutropenic model of disseminatedCandidainfection for detection of 65-kDa protein.For determination of circulating antigens in an experimental model of candidemia, 6-week-old, 22- to 24-g BALB/c female mice (n⫽21) were rendered neutropenic (absolute neutrophil count of⬍100/ml) by administration of 150 mg/kg of intraperitoneal (i.p.) cyclophosphamide 4 days prior to infection. This was followed by a second dose of 100 mg/kg i.p. cyclophosphamide 1 day prior to
infection and then 100 mg/kg i.p. cyclophosphamide every other day thereafter. Twenty-four hours after the second dose of cyclophosphamide, three groups of mice (n⫽7/group) were intravenously inoculated with 1⫻106cells ofC.
albicans, 1⫻106cells ofC. parapsilosis, or 1⫻106cells ofC. tropicalis. A pool
of serum was collected daily for each group for up to 7 days or until the group died. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Federal University of Sa˜o Paulo.
Human clinical samples.A total of 20 candidemic patients, diagnosed by a positive blood culture processed by the Bactec 9240 system, were sequentially enrolled in this study. Volunteers were selected among patients who signed the informed consent form and were admitted to the Hospital Sa˜o Paulo, Sa˜o Paulo, Brazil, between February 2006 and July 2007. Serum samples for antigen detec-tion were obtained at the time of diagnosis of candidemia and after different intervals. Stocked serum samples collected from candidemia patients before the onset of fungemia were also evaluated when available. A pool of serum samples from healthy volunteers (n ⫽20, blood donors) were included as negative controls. To verify cross-reactions, sera from patients with active histoplasmosis (n⫽3), active paracoccidioidomycosis (n⫽3), active Jorge Lobo’s disease (n⫽
3), and active aspergillosis (n⫽3) were also tested.
RESULTS
Composition of the immunodominant antigen.
After
SDS-PAGE of the exoantigens from
C. albicans
,
C. parapsilosis
,
C.
tropicalis
, and
C. glabrata
, a common protein band of 65 kDa
was found among the three species
C. albicans
,
C. tropicalis
,
and
C. parapsilosis
.
C. glabrata
did not show this molecule (Fig.
2). In the pilot study, the rabbit anti-
C. albicans
hyperimmune
serum recognized the 65-kDa antigen present in the
exoanti-gens of
C. albicans
,
C. tropicalis
, and
C. parapsilosis
by
immu-noblotting.
were produced to detect the 65-kDa protein. A panel of
dif-ferent hybridoma lines reactive to
C. albicans
65-kDa antigen
was obtained. MAbs belonged to the IgG1 subclass and
rec-ognized an antigenic determinant of
C. albicans
with a relative
mass of 65 kDa by Western blotting (Fig. 3). This MAb also
recognized the 65-kDa molecule present in
C. tropicalis
and
C.
parapsilosis
. This MAb showed no reactivity with exoantigens
of
H. capsulatum
,
S. schenckii
,
T. rubrum
,
A. fumigatus
, and
C.
glabrata
and was selected to develop the inh-ELISA test. This
MAb was designated “IgG1-2.”
Dynamics of 65-kDa antigenemia in a murine model of
in-vasive candidiasis by MAb inh-ELISA.
The inh-ELISA was
first tested in a murine model of invasive candidiasis before
validation in the human system. Three groups of mice were
infected with
C. albicans
,
C. tropicalis
, and
C. parapsilosis
,
re-spectively. Animals were not challenged with
C. glabrata
be-cause the 65-kDa protein was not detected in this species. All
animals presented the 65-kDa protein in serum samples
col-lected daily for up to 7 days. In all three groups, the final serum
sample collection occurred when the infected mice were dead.
In the murine model infected with
C. albicans
, the 65-kDa
molecule was detected in the range of 0.019 to 2.6
g/ml; in the
C. tropicalis
model, the molecule was detected in the range of
0.012 to 3.25
g/ml; and in the
C. parapsilosis
model, the
molecule was detected in the range of 0.019 to 1.68
g/ml.
Figure 4 shows typical curves obtained from the groups of
animals infected with different
Candida
species. Antigen
con-centrations higher than 0.23
g/ml (maximum concentration
obtained with 10 normal mouse sera) were considered positive.
Dynamics of 65-kDa antigenemia in patients with invasive
candidiasis.
Figure 5 illustrates the standard inhibition curve
FIG. 2. SDS-PAGE showing the presence of the 65-kDa protein in
exoantigens from
C
.
tropicalis
(lane 1),
C. parapsilosis
(lane 2), and
C.
albicans
(lane 3) and its absence in
C. glabrata
(lane 4). P, molecular
mass standard.
FIG. 3. Reactivity of the MAb specific for the 65-kDa protein of
C.
albicans.
The MAb recognized 65-kDa proteins of
C. albicans
(lane 9),
C. parapsilosis
(lane 8), and
C. tropicalis
(lane 7). Lanes: 1,
H.
capsu-latum
; 2,
A. fumigatus
; 3,
S. schenckii
; 4,
P. brasiliensis
; 5,
T. rubrum
; 6,
C. glabrata
; P, molecular mass standard.
FIG. 4. Detection of circulating antigens in murine candidemia
models infected with
C
.
tropicalis
(A),
C
.
parapsilosis
(B), and
C
.
albi-cans
(C) using a MAb. The concentration of circulating antigens in
normal mouse serum was 0.23
g/ml (maximum concentration
ob-tained with 10 normal mouse sera). Dots represent a pooled medium
made up of seven sera.
FIG. 5. Standard inhibition curve of MAb against the 65-kDa
pro-tein of
C. albicans
constructed with known quantities of purified
65-kDa protein.
obtained with known quantities of
C. albicans
65-kDa protein.
This curve was used to determine the concentration of 65-kDa
antigen in each sample tested. Antigenemia was present in 20
candidemia patients, from 10 to 87 years of age (median, 47.1
years): 45% were male, and all had been exposed to multiple
risk factors before developing candidemia. All patients were
treated with antifungal drugs 1 to 4 days after the incidence of
candidemia. A total of 16 of the 20 candidemic patients
exhib-ited the 65-kDa protein in concentrations ranging from 0.072
concentrations in each patient serum and the species of
Can-dida
isolated from blood cultures with the Bactec 9240 system.
DISCUSSION
During pilot studies analyzing exoantigens from the more
prevalent
Candida
species, isolated from Brazilian patients
with candidemia, we found an intense 65-kDa molecule
com-mon to
C. albicans
,
C. tropicalis
, and
C. parapsilosis
. We
hy-pothesized the presence of this molecule could be used as a
marker of invasive candidiasis. The 65-kDa molecule from
C.
albicans
was used to produce a hyperimmune serum (in
rab-bits) that was able to recognize this antigen in
C. albicans
,
C.
tropicalis
, and
C. parapsilosis
blot tests. To verify the efficiency
of detection of the 65-kDa antigen in the inh-ELISA system,
the system was tested in a murine experimental model of
dis-seminated candidiasis before its application in a human
sys-tem. The results showed detection of this antigen was possible
in murine candidiasis evoked by the three species of
Candida
.
To increase the sensitivity of the inh-ELISA test, MAb
against 65-kDa
C. albicans
was produced and tested in
C.
albicans
,
C. tropicalis
, and
C. parapsilosis
murine models of
disseminated candidiasis infection. During the experiments,
the 65-kDa antigen was detected in concentrations ranging
from 0.012 to 3.25
g/ml of serum and the inh-ELISA was
sensitive enough to detect 100% of the sera tested. The
dy-namics of 65-kDa antigenemia was different in each group; but
in each group, “clearance” of the 65-kDa antigen was observed
in some serum samples collected after infection and the
clear-ance was not dependent on antifungal treatment. The drop in
the level of 65-kDa antigen in mice infected with
C. tropicalis
does not necessarily indicate the mice were cleared, because on
the last day sample sera were collected, the group of mice was
dead. The levels in control sera from uninfected animals were
always considered low.
Once the efficiency of antigen detection was established in
murine models with anti-65-kDa MAb, sera from invasive
can-didiasis patients from the ICU were collected for antigen
de-tection. These serum samples were collected at the moment
when the Bactec 9240 blood culture system indicated the
pres-ence of yeasts and also during subsequent days. When possible,
samples were collected before the onset of candidemia.
A total of 20 patients suspected of candidemia were studied,
and 16 (80%) patients were positive for the presence of the
65-kDa antigen, in concentrations ranging from 0.07 to 5.0
g/ml. Three dynamics of 65-kDa antigenemia were observed
among these patients. Four patients experienced a total
clear-ance of 65-kDa antigenemia after treatment with antifungal
drugs: the concentration of 65-kDa antigen in the last sample
tested was less than 0.11
g/ml. Five patients experienced an
initial clearance after treatment with antifungal drugs (with the
exception of patient 1) and a relapse of antigenemia (patient 7
presented an initial clearance, a relapse of antigenemia, total
clearance of antigenemia, and survival after treatment). Seven
patients experienced a partial clearance of antigenemia in
which the concentration of 65-kDa antigen in the last sample
analyzed was greater than 0.25
g/ml. Patients with persistent
65-kDa antigenemia after antifungal treatment did not survive,
with the exception of patient 20. During patient follow-up,
some sequential sera showed “clearance” of the antigen which
may be explained by rapid renal clearance or the host’s
im-mune response. Otherwise, the inh-ELISA was able to detect
the 65-kDa protein in serum samples collected before the
onset of candidemia, indicating its possible use for early
diag-nosis of disseminate candidiasis. Although five species of
Can-dida
—
C. albicans
,
C. parapsilosis
,
C. tropicalis
,
C. kefyr
, and
C.
glabrata
—were isolated from the blood of these patients, the
65-kDa antigen was detected only in sera from those patients
infected with
C. albicans
,
C. parapsilosis
, or
C. tropicalis
.
It is extremely complex to evaluate the cause of mortality in
patients with candidemia. Considering all underlying
condi-tions present at the moment a patient develops candidemia, it
is possible a significant proportion of patients will have
mor-tality related to candidemia but not attributable to it.
Conse-quently, it is hard to establish a direct connection between
antigen levels and risk of mortality. Data obtained from animal
models suggest that transient clearance of antigen from blood
may occur in animals that will die of systemic infection. In
humans, no positive results were found among the sera from
healthy people.
Available data suggest the inh-ELISA has good sensitivity
and specificity for the diagnosis of candidemia. However, more
data are needed to clarify whether this test may or may not be
used for monitoring clearance of
Candida
infections from
blood or tissues. Based on our preliminary data, negative
inh-ELISA results were not correlated with survival. In addition,
we observed transient clearance of antigenemia may occur with
further increase of antigen after an interval. Taken together,
this suggests the level of antigen in serum may not correlate in
time with the burden of infection.
Hypothetically, the clearance can be explained either by the
immune response of the host, where phagocytes ingest the
molecule of 65-kDa antigen, decreasing the levels in the serum
of patients at the moment of sample collection, or by filtration
of blood in the kidneys, where the molecule could also be
TABLE 1. Antigen detection by inh-ELISA in sera from patients
with candidemia at the moment of diagnosis
Patient Concn (65-kDa proteing/ml) ofa Species isolated fromblood culture
1
2.24
C. tropicalis
2
ND
C. kefyr
3
ND
C. glabrata
4
ND
C. albicans
5
0.81
C. albicans
6
ND
C. glabrata
7
2.56
C. parapsilosis
8
ND
C. albicans
9
1.87
C. albicans
10
1.83
C. tropicalis
11
ND
C. albicans
12
ND
C. albicans
13
1.25
C. albicans
14
5
C. albicans
15
1.3
C. tropicalis
16
1.5
C. albicans
17
1.28
C. tropicalis
18
1.3
C. tropicalis
19
1.75
C. albicans
20
1.98
C. parapsilosis
aShown is the concentration of 65-kDa protein in sera from patients at the moment of diagnosis (Bactec positive). ND, not detected.
eliminated. However, in this study it was not possible to collect
urine from our patients because they were in the ICU with
renal failure. In the future, we will do this analysis.
In this study, some patients presented with the 65-kDa
an-tigen in sera collected before the onset of candidemia
(diag-nosed by the Bactec system). Therefore, in patients suspected
of disseminated candidiasis, we suggest serum samples be
col-lected as early as possible in an attempt to detect this marker
antigen before candidemia is diagnosed by the Bactec system.
Early diagnosis of candidemia would allow for appropriate
antifungal treatment and prevention of
Candida
dissemination,
reducing the rate of mortality among these patients. Blood
cultures are still considered the “gold standard” for the
diag-nosis of candidemia, as no single molecular method has been
proved superior. This may be confirmed by a recent review of
the criteria for diagnosis of invasive fungal infections published
by NIH (2).
In conclusion, we propose the use of this inh-ELISA system,
using a species-specific MAb, as an additional test for
detec-tion of circulating antigen in patients with candidemia. The use
of the 65-kDa antigen detection system described here will
provide a rapid and specific means of diagnosis. Moreover, this
test has the potential to follow the course of antimycotic
ther-apy by monitoring the reduction in fungal load, as evidenced by
sequential reduction in antigen detection. This will be
ad-dressed in future studies.
ACKNOWLEDGMENT
This work was supported by a grant from FAPESP and CNPq.
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