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Journal of Clinical Microbiology, January 2000, p. 438-443, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Aspergillus fumigatus Antigen Detection
in Sera from Patients at Risk for Invasive Aspergillosis
Bernabé F. F.
Chumpitazi,*
Claudine
Pinel,
Bernadette
Lebeau,
Pierre
Ambroise-Thomas, and
Renee
Grillot
Département de Parasitologie-Mycologie
Médicale et Moléculaire, UPRES A, CNRS 5082, Hôpital Albert Michallon, 38043 Grenoble, France
Received 18 February 1999/Returned for modification 16 August
1999/Accepted 15 September 1999
 |
ABSTRACT |
We have developed an inhibition enzyme immunoassay (inhibition-EIA)
to monitor for the occurrence of invasive aspergillosis (IA) in sera
from 45 immunocompromised (IC) patients. The test uses rabbit
polyclonal antibodies and a mixture of components from
Aspergillus fumigatus, containing three predominant
antigens with molecular weights of 18,000, 33,000, and 56,000. Circulating antigens were found in five of seven proven cases of IA due
to A. fumigatus. In two of the five positive cases,
antigenemia was detected with inhibition-EIA earlier than with X ray or
other biological methods. No antigens were detected in the sera from two patients with proven IA due to Aspergillus flavus and
Aspergillus terreus nor in the sera from four patients with
probable IA. Circulating antigens were not detected in the control
group, composed of 30 healthy adult blood donors. Four of the 32 at-risk patients examined, though they displayed no definite evidence
of IA, gave a positive result in this test. The sensitivity,
specificity, and positive predictive value of inhibition-EIA were 71.4, 94.4, and 71.2%, respectively. The data were compared with those
obtained by a latex agglutination assay of galactomannan (GM) that was
positive in only one patient with probable IA. The higher sensitivity
obtained by inhibition-EIA may well be due to its ability to detect
circulating antigens other than GM in the sera of IC patients with IA.
Detecting these antigens may improve the diagnosis of IA, as they may
serve as markers of this infection.
| |
TEXT |
Invasive aspergillosis (IA) has been
a significant cause of life-threatening opportunistic infections in
immunosuppressed hosts (9). The incidence of IA, which is
the second most common cause of fungal infection in this type of
patient, varies from 0.5 to 25% (10, 17, 30, 38, 42). The
reported mortality mainly varies from 50% to nearly 100% (9, 10,
22, 24, 38). The diagnosis is consequential, since an early
diagnosis combined with adequate therapy may improve the clinical
outcome in immunosuppressed patients (1, 6). However,
establishing the diagnosis continues to be a major problem for the
clinician, since the clinical symptoms of IA are not pathognomonic of
the disease, while histological and culture confirmations are often difficult to obtain antemortem (8, 15). Moreover, the
efficient techniques of imaging do not always allow adequate
discrimination among the different etiologies involved in this type of
symptoms. Furthermore, the typical form of serological evidence, that
is, increased antibody levels, is usually not revealed in this type of
patient. The detection of circulating Aspergillus antigens and detection of Aspergillus DNA (35, 44) are two
of the most promising methods to diagnose IA in at-risk patients. Many
studies report the detection of circulating antigens (11, 12, 14, 21, 28, 29, 34-37, 41, 43, 46). A commercially available test,
Pastorex Aspergillus (Sanofi Diagnostic Pasteur,
Marnes-la-Coquette, France), can be very specific but appears to be
relatively insensitive (45). In this study, we did not
systematically use the Platelia Aspergillus kit, since it is
more sensitive but less specific than the Pastorex system (5, 39,
40). Moreover, a recent study has suggested that
heat-resistant galactomannan (GM) is not eliminated by the
processes of food sterilization and may reach the circulation
through damaged intestinal mucosa and cause false-positive results in
tests to detect antigenemia (25). Therefore, in an effort to
improve the diagnosis of IA, an inhibition enzyme immunoassay
(inhibition-EIA) developed in our laboratory was selected for
investigation. This system, which is thought to mainly detect antigens
with Mrs of 18,000, 33,000, and 56,000, was
compared to the Pastorex Aspergillus test for the detection of GM. The results obtained in each case were related to the clinical data.
Case definitions.
IA, associated with an immunodebilitated
condition (i.e., prolonged neutropenia for at least 10 days within the
previous 2 months, immunosuppressive therapy within the last month, or
a previous episode of fungal infection) and with persistent fever (>38°C) for at least 3 days, despite a broad-spectrum
antibiotherapy, was diagnosed mainly by direct isolation and
culture of the organism from bronchopulmonary specimens and biopsies
obtained by a sterile procedure (15). Additional diagnostic
criteria included radiological disturbances (i.e., abnormal
characteristic signs on chest radiography consistent with infection)
obtained by the effective techniques of imaging or computed tomography.
Group I.
In the context defined above, proven IA was diagnosed
by histologic evidence of the presence of hyphae in tissue specimens and in vitro growth of Aspergillus species in culture.
Group II.
Probable IA cases were defined as demonstrating at
least one criterion from the context section and one major or two minor clinical criteria from an abnormal site consistent with infection and
as presenting one of the following criteria: hyphae in fiber-endoscopic samples, positive Aspergillus culture from bronchoalveolar
lavage fluid or bronchial aspirates, and testing positive for
antigenemia with Pastorex Aspergillus.
Group III.
Patients who were at risk for IA had only clinical
evidence of infection. Possible IA cases were defined as meeting at
least one criterion from the context section and one microbiological or
clinical criterion of infection as cited above.
In the present study, tests positive for antigenemia which were
obtained by using inhibition-EIA were not considered in classifying patients, since this method was under evaluation.
Patients.
Group I contained nine patients (three women and six
men, ranging from 26 to 60 years of age) with proven IA (Table
1). Group II contained four patients (all
men, ranging from 62 to 72 years of age) infected by A. fumigatus, with probable IA (Table 1). Group III contained 32 patients (16 women and 16 men, ranging from 10 to 76 years of age) at
risk for IA (in this group, 26 patients had hematological disorders,
two had received liver transplants, and four had various pathologic
disorders associated with a severely immunodebilitating condition
[i.e., myeloma and cardiac disease]). The sera were obtained from all
patients during a retrospective and prospective longitudinal study (up
to 65 weeks).
Control group.
This group comprised 30 healthy adult blood
donors (28 women and 2 men, ranging from 19 to 36 years of age) without
specific antibodies to A. fumigatus in their sera, as
determined by enzyme immunoassay (EIA), immunofluorescent antibody test
(IFAT), and counterimmunoelectrophoresis (CIE).
Antigens.
Aspergillus fumigatus antigens from a
Longbottom strain (NCPF 2109) were prepared in Panmede medium (Paines
and Byrne, Greendford, United Kingdom) and were grown in a stationary
3-week culture at 27°C (CF27), 37°C (CF37), and 42°C (CF42)
(31). Briefly, the mycelium was broken in the culture
medium; the suspension was filtered, dialyzed, and concentrated in
Amicon membrane (PM10); and was finally lyophilized. The antigens were
stored at 4°C until required.
Rabbit antisera.
Antisera to CF27, CF37, and CF42 were raised
in female New Zealand White rabbits. Ten milligrams of lyophilized
antigens in 0.9% NaCl (wt/vol) was mixed with an equal volume of
Freund's complete adjuvant and was injected intradermally at multiple
sites. Two weeks later, booster injections of each antigen, to which Freund's incomplete adjuvant had been added, were administered every 2 weeks over a period of 1 to 2 months until optimum antibody levels were
detected by an EIA. Sera obtained from rabbits before immunization were
used as negative controls.
SDS-PAGE and immunoblotting.
The procedure described elsewhere
(7) was adapted to Aspergillus antigens. Briefly,
20 mg of lyophilized CF37 in 1 ml of a solution containing 0.25 M Tris
(pH 6.8), 0.25 M EDTA, 5% sodium dodecyl sulfate (SDS), and 0.05%
-mercaptoethanol was denatured for 3 min at 100°C. Following
SDS-polyacrylamide gel electrophoresis (PAGE) of CF-37, using a
separating gel of 12.5% (at 250 V for 45 min at 15°C), the gel was
blotted with nitrocellulose sheets (NC), which were then fixed by
drying at 37°C for 15 min. After saturation of the free binding sites
on NC, antisera from rabbits were used at dilutions of 1:100 and 1:200
and were added to the NC at 37°C for 1 h. After four 2-min
washes in phosphate-buffered saline (PBS), goat anti-rabbit
immunoglobulin G (IgG) (1:1,500)-alkaline phosphatase conjugate (Miles,
Puteaux, France) was added to the NC, and the mixture was incubated at
37°C for 1 h. After a new cycle of washing, the bands were
visualized with 0.15 mM nitroblue tetrazolium plus 0.15 mM
5-bromo-4-chloro-3-indolylphosphate. The reaction was stopped by two
5-min washes in distilled water.
Human antibodies to A. fumigatus.
Antibodies were
detected by EIA (IgG), IFAT (IgG and IgM), and CIE (19).
Readings higher than the following cutoffs were considered positive:
0.44 (EIA), 1:80 (IFAT), and two arcs of precipitation (CIE).
Circulating antigen detection by inhibition-EIA.
One hundred
milliliters of antigens (2 µg of lyophilized CF37/ml) in a solution
containing 50 mM carbonate buffer, 25 mM EDTA, and 0.02%
NaN3 (pH 9.6) was used to coat polystyrene microtiter plates (M129B; Dynatech, France) overnight at 4°C.
Two aliquots of test human serum (50 µl each) were diluted 1:5 in 145 mM PBS containing 5% nonfat milk, 0.01% Triton X-100,
25 mM EDTA, and
0.02% NaN
3 (pH 7.2); one aliquot was heated at
80°C for
30 min while the other aliquot remained unheated. Antiserum
from rabbit
to CF27
A. fumigatus antigens and preimmune, negative
serum
were diluted 1:1,000 in the same solution as used above,
then mixed
with equal volumes of human sera, in both heated and
unheated samples.
The mixture was incubated at 4°C
overnight.
The antigen-coated plates were blocked with a solution containing 145 mM PBS and 5% nonfat milk (pH 7.2) at 37°C for 15 min
and then
exposed to preincubated human and rabbit sera for 30
min at
37°C. After four 2-min washes in a solution containing
145 mM PBS and
0.01% Triton X-100, 100 µl of goat anti-rabbit
IgG (1:1,500)
peroxidase conjugate (CALTAG; TEBU, Le Perray en
Yvelines, France)
diluted in the solution of 145 mM PBS (pH 7.2),
5% nonfat milk, and
0.01% Triton X-100 were added to each well,
and the mixtures were
again incubated at 37°C for 30 min. After
a new cycle of washing,
absorbance at 492 nm (
A492) was measured
following incubation for 10 min in a solution of
ortho-phenylenediamine-2HCl
(3 mg/ml) and
H
2O
2 (0.08%) in citrate buffer (pH 5.15), and
the
reaction was stopped with 2 N H
2SO
4.
Circulating-antigen levels
were estimated from a standard calibration
curve plotting percentage
of inhibition of antibody binding (% inh)
versus amount of CF37
(0, 4, 40, 400, and 4,000 ng/ml). The inhibition
cutoff values
of 22.9% and 34.9%, for heated and unheated human sera,
respectively,
were determined from the
A492 of
samples from the control group
by using the average reading plus twice
the standard deviation.
The % inh was calculated by using the
following equation: % inh
= (1
Ax/Ac) × 100, where Ax is
the difference in
A492 between
the positive and
negative rabbit sera in the presence of the test
human serum "x"
and Ac is the difference in
A492 between the
positive
and negative rabbit sera in the presence of the pool of human
sera from control group "c".
To detect circulating antigens of
A. fumigatus, the
parameters of inhibition-EIA were established by titration of CF37
antigen,
goat anti-rabbit IgG peroxidase conjugate, and rabbit
hyperimmune
serum to CF27, CF37, and CF42 in the presence of a known
amount
of added antigen (Fig.
1). Optimal
results were obtained with
CF37 for coating the plates at 2 µg/ml,
the peroxidase conjugate
at 1:1,500, and the anti-CF27 rabbit serum at
1:1,000.
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FIG. 1.
Optimization of the parameters of inhibition-EIA. Three
rabbit hyperimmune sera were tested: anti-CF27, anti-CF37, and
anti-CF42 (1:1,000). In this case, the plate was coated overnight with
CF37 antigen at 2 µg/ml, and peroxidase conjugate was used at
1:1,500. The highest % inh was obtained experimentally with
anti-CF27.
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|
SDS-PAGE and Western blotting were carried out in order to determine
which antigens were detected by inhibition-EIA. The mixture
of antigens
used for coating the plates contained major antigens
with the following
Mrs: 62,000, 56,000, 50,000, 42,000, 33,000,
and
18,000 (Table
2). The anti-CF27 rabbit
serum used in the
inhibition-EIA mainly recognized
Mrs of 56,000, 33,000, and 18,000.
It is
therefore probable that the inhibition-EIA, as used in this
study,
mainly detected these three antigens.
The antigens of
A. fumigatus used for coating the plates in
inhibition-EIA were positive in the Pastorex
Aspergillus
test
at a 1:64 dilution. If the highest sensitivity of this test is
assumed to occur (
2), this dilution contains approximately
15 ng of GM per
ml.
Circulating antigens of
A. fumigatus were detected by
inhibition-EIA only when heated serum samples from patients were used
(Table
3). It is interesting to note that
in the six samples
obtained from patient number 1, negative inhibitions
of 18 to
41.5% were observed when unheated sera were tested. In
positive
cases (Table
3), the antigen detection usually gave
fluctuating
levels of antigenemia in sera from immunodebilitated
patients
with IA (Fig.
2). However, in
patients 3 and 8, all six samples
were positive with a % inh between
27.4 and 40.3%. In two cases,
antigen detection was earlier than with
X ray or biological methods
(Fig.
2). However, one case was diagnosed
10 days later, and the
other two were diagnosed at the same time. In
addition, the %
inh due to circulating antigens varied from 24.9% to
40.3% (110
to 180 ng/ml) when human sera were heated before incubation
with
rabbit hyperimmune serum. If the data in group I are taken into
account, the sensitivity, specificity, and positive predictive
value of
inhibition-EIA were 71.4, 94.4, and 71.2%, respectively.
In group II,
the sera from patients with probable IA were negative
by
inhibition-EIA, but one serum sample out of three was positive
with the
Pastorex
Aspergillus test (in this group the sensitivities
were 0% and 25% [
n = 4], respectively). In all
other cases, this
last test failed to detect any circulating antigens
in patients'
sera.
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FIG. 2.
Detection of antigen in a patient (number 2) with proven
IA (in trachea, vertebra, and brain) in terms of number of days after
initial detection. Positive cultures were found first in trachea (first
arrow), then in brain (second arrow), and finally in vertebra (third
arrow). In this case, antigenemia detected (+) in heated sera was
transient and repeated. It was positive at 4 of 13 time points and in
all of the earlier stages (up to 60 days). The detection of circulating
antigens in several samples may be necessary. Unheated serum samples
gave negative results. The error bars represent standard deviations.
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|
In group III, of the four cases positive by inhibition-EIA (Table
3),
two were related to the transient presence of
A. fumigatus in the trachea and in the bronchoalveolar lavage. In the third
case,
the patient had significant variations in the levels of
antibodies to
A. fumigatus. In the fourth case, the fungus was
not found
either on direct examination or in cultures. Therefore,
the two first
cases may be considered as possible IA, although
only minor pathology
was
observed.
Aspergillus antigens were not detected by inhibition-EIA in
the sera of patients with proven IA who were infected with
Aspergillus terreus and
Aspergillus flavus.
No false positive cases were obtained with the Pastorex
Aspergillus test in any
group.
In the present study, antigenemia detection by inhibition-EIA could be
used as a method to diagnose and monitor IA in patients
at risk for
aspergillosis. This is supported by the data obtained
from proven and
suspected IA cases. However, other methods can
be used in other
instances to diagnose the disorder more quickly,
as mortality may be
reduced with adequate, timely therapy. In
probable cases of IA,
antigenemia was not detected by inhibition
EIA, and this could be an
inconvenience of this method. In group
III, inhibition EIA may be
useful, since it may aid in the diagnosis
and monitoring of possible
cases of IA after antigenemia is detected
in suspected patients, as was
the case for two patients in our
study with transient presence of
A. fumigatus in the trachea and
in the bronchoalveolar
lavage.
In this study, circulating antigens of
A. fumigatus detected
by inhibition-EIA were most likely those with
Mrs of 56,000,
33,000, and 18,000, since rabbit
serum anti-CF27 mainly recognized
these three components on Western
blots. Therefore, EIA with CF37
and anti-CF27 may detect antigens with
Mrs of 56,000, 33,000,
and 18,000. The
56,000-
Mr antigen of CF37 may be the same as the
one described by Framatico and Buckley (
13) who found that
sera
from patients with IA reacted against an antigen with an
Mr of
58,000. In that study, Framatico and
Buckley noted that this antigen
may also be found in culture medium. In
addition, Reichard et
al. (
33) characterized an
extracellular serine proteinase from
A. fumigatus with an
Mr of 32,000 to 33,000 which might be one
of the
components used for coating the plates in the present study.
Moreover,
Haynes et al. (
18) detected two major antigens with
Mrs of 18,000 and 11,000 in serial urine samples
from patients
who developed IA. Latgé et al. (
23)
described the antigen with
an
Mr of 18,000 as
being secreted and present in the urine of
patients with IA. In the
present study,
Mr determination suggests
that
this antigen with an
Mr of 18,000 may also be
part of the
antigens detected by inhibition-EIA. Thus, this test may
detect
several antigens at the same time, which might explain the
greater
sensitivity of the test compared to the latex agglutination
assay
based solely on GM
detection.
Theoretically, anti-CF37 seemed the most appropriate reagent for our
test, but this proved not to be the case. This discrepancy
may be due
to the immunological or cytotoxic (
3,
26) properties
of the
CF37 antigen which was involved in down regulation of anti-CF37
levels
in rabbits after the last boost (data not shown). Moreover,
this
antigen is rich in chymotrypsin and catalase (data not shown).
In
addition, GM is also a component of CF37, as demonstrated by
the
Pastorex
Aspergillus test but not by Western blotting, since
this method can detect proteins or glycoproteins. Even if GM is
a
component of CF37, the inhibition-EIA plates were coated with
approximately 1 ng of this macromolecule per ml, which is the
limit of
detection of a sensitive EIA (
5,
39,
40). As CF37
is
composed of several major antigens, GM is unlikely to be detected
by
the inhibition-EIA. This hypothesis is supported by the fact
that the
one serum sample which was positive in the Pastorex
Aspergillus test was negative by inhibition-EIA.
In all of the other proven IA cases, only inhibition-EIA gave a
positive result. In order to confirm the absence of GM from
the sera of
these patients, a sensitive EIA (Platelia) was used
retrospectively in
serum samples from five cases of proven IA,
and only one serum sample
was found to be positive by this test
(data not shown). The fact that
inhibition-EIA was more sensitive
than GM detection may be explained by
the multiple targets and
by the fact that GM antigens are rapidly
removed from circulation
by the formation of immune complexes and by
receptor-mediated
endocytosis by Kupffer's cells in the liver
(
4). The low level
of antigen detection is also thought to
be due to fluctuating
levels of antigenemia (
35,
39,
40).
These fluctuations have
also been observed in the present study with
CF37 antigens of
A. fumigatus.
In the present study, the sensitivity of the assay is good enough,
given that IA was diagnosed despite the heterogeneous underlying
diseases of these patients. However, serum components and immune
complexes can interfere with the detection of circulating antigens
by
inhibition-EIA, since antigens were detected in the samples
only after
heat treatment. Many laboratories use only heated serum
for detecting
these antigens (
11,
29,
35,
39,
41,
44,
46). This may
indicate that heat-sensitive molecules present
in human sera are
responsible for this phenomenon. The relationship
between circulating
antigens and these molecules may be specific,
e.g., like specific
antibodies, or not specific, e.g., like protein-protein
interactions.
The test used in this study appears to be more specific for
A. fumigatus, since it did not recognize antigens in the serum
samples from group I patients infected with
A. flavus and
A. terreus.
This might be regarded both as an inconvenience
and as an advantage
in diagnosing IA, since the most prevalent species
is
A. fumigatus.
In the present study, antigenemia was detected the earliest in two
cases of proven IA, but the antigenemia was transient in
these patients
at that time, and its detection required the testing
of several
samples. The sensitivity of this test may increase
when heated serum is
used. However, in IA diagnosis, the antigen
sensitivity may also be
improved by determining the presence of
antigenuria (
2), by
detecting antigens in cerebrospinal fluid
(
32), or by
combining other methods (
6,
16,
20,
27,
32,
39,
40). Yet,
given the diversity of circulating antigens,
the different methods seem
to be complementary for diagnosing
IA in immunocompromised patients.
However, improving the sequencing
of purified antigens and the
identification of epitopes with high
specificity for IA diagnosis is
the next step in optimizing the
method. Finally, even though the
detection of a wide variety of
antigens in patients' sera is not
always proof of IA, as was indicated
by the data of group III, the
antigen data should be taken into
account in the monitoring of patients
at risk for
IA.
| |
ACKNOWLEDGMENTS |
We thank V. M. Hearn, S. Picot, and S. Durville for scientific
comments and/or assistance with the preparation of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Département de Parasitologie-Mycologie, Hôpital Albert
Michallon, BP 217, 38043 Grenoble cedex 9, France. Phone: (33) 4 76 63 71 01. Fax: (33) 4 76 51 86 67. E-mail:
Bernabe.Chumpitazi{at}ujf-grenoble.fr.
| |
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Journal of Clinical Microbiology, January 2000, p. 438-443, Vol. 38, No. 1
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