Galactomannan in Nonserum Specimens • CID 2004:39 (15 November) • 1467
R E V I E W A R T I C L E
Utility of Aspergillus Antigen Detection in Specimens
Other than Serum Specimens
Rocus R. Klont, Monique A. S. H. Mennink-Kersten, and Paul E. Verweij
Department of Medical Microbiology, University Medical Center St. Radboud, Nijmegen University Center for Infectious Diseases, Nijmegen,
The detection of circulating galactomannan in serum is an important tool for the early diagnosis of invasive
aspergillosis. A commercial enzyme-linked immunosorbent assay (Platelia Aspergillus; BioRad) was shown to
be both highly sensitive and specific for detection of galactomannan in serum samples. Despite the fact that
this assay is validated for serum samples, specimens of other body fluids are increasingly used for detection
of galactomannan, including urine, bronchoalveolar lavage fluid, and cerebrospinal fluid. Review of the lit-
erature shows that galactomannan can be detected in each of these samples from patients with invasive
aspergillosis with higher sensitivity than is the case with culture, as well as early in the course of infection.
However, the evidence thus far is based on case reports—predominantly retrospective studies—that often
include heterogeneous patient populations and limited numbers of cases of proven infection. Clearly, well-
designed prospective studies with systematic sampling and use of consensus case definitions are needed to
compare the performance of antigen detection in samples other than serum specimens with that in serum
Invasive aspergillosis is the most common opportun-
istic invasive mycosis . This disease is mainly caused
by Aspergillus fumigatus . Invasive aspergillosis pre-
dominantly affects patients with neutropenia or whose
neutrophils are functionally compromised. The inci-
dence of invasive aspergillosis has increased because of
the increased number of patients undergoing hema-
topoietic stem cell transplantation or receiving courses
of corticosteroid therapy for a prolonged time . In
addition, invasive aspergillosis may affect solid organ
transplant recipients . Despite the development and
registration of new antifungal drugs, such as caspofun-
gin and voriconazole, the overall survival rate remains
low (30%–50%) [5, 6]. This is partly because of the
difficulty in establishing the diagnosis in an early stage
of the infection. Early diagnosis and subsequent early
Received 25 April 2004; accepted 23 July 2004; electronically published 26
Reprints or correspondence: Dr. R. R. Klont, Dept. of Medical Microbiology,
University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The
Clinical Infectious Diseases 2004;39:1467–74
? 2004 by the Infectious Diseases Society of America. All rights reserved.
initiation of therapy improves outcome [5, 7, 8]. How-
ever, clinical signs and symptoms are generally non-
specific, and characteristic lesions are frequently absent
from chest radiographs of neutropenic patients. The
use of high-resolution CT with high-risk patients could
result in earlier diagnosis, but characteristic lesions,
such as the halo sign and the crescent sign, are not
specific for Aspergillus species. The reference standard
for diagnosis, histopathologic examination and subse-
quent culture of the tissue samples, is often not done
because the patient’s status prohibits invasive proce-
dures (e.g., the patient has thrombocytopenia or poor
clinical condition). Furthermore, cultures for fungi and
cytopathological examination of respiratory specimens
often yield negative results and lack sensitivity for de-
tecting the fungus in an early stage of the infection .
Because of the limitations of the aforementioned di-
agnostic methods, a nonculture method, based on the
detection of the Aspergillus antigen galactomannan,has
Galactomannan is a cell wall polysaccharide that is
released by Aspergillus species during growth. A com-
mercially available sandwichELISA(PlateliaAspergillus;
BioRad) detects galactomannan by use of a rat mono-
1468 • CID 2004:39 (15 November) • Klont et al.
clonal antibody (EB-A2), which is used as both detector and
captor. This antibody reacts with the b(1r5)-linked galacto-
furanosyl residues that constitute the side chains of galacto-
mannan . The test has been validated for serum specimens
only and has a detection limit of ∼1 ng/mL.Reportedsensitivity
and specificity range from 50% to 92.6% and from 94% to
99.6%, respectively, in patients with hematologic malignancy
[11–15]. Reported positive and negative predictive values for
patients with proven invasive aspergillosis range from 85% to
93% and 95% to 98.7%, respectively [11–15]. False-positive
results have been reported in adults and range from 5.7% to
14% when serum samples are used, but this may be overesti-
mated because of diagnostic uncertainty [11, 12, 16, 17]. Rates
of false-positive results are higher among pediatric patientsand
neonates and may be as high as 83% [13, 17, 18]. Reasons for
false reactivity remain largely unknown, although recently, pi-
peracillin-tazobactam was shown to cause cross-reactivity in
adults [19, 20], and cross-reacting epitopes from Bifidobacter-
ium species were proposed as a cause in neonates . Issues
regarding causes of false reactivity were recently reviewed else-
Circulating galactomannan may be detected at a median of
5–8 days (range, 1–27 days) before clinical signs and symptoms
of invasive aspergillosis become evident [11–13, 23]. Further-
more, the concentration of circulating galactomannan corre-
sponds with the fungal tissue burden[17,24]andmaytherefore
be used to monitor the patient’s response to antifungal treat-
ment [12, 23]. Other methods to detect galactomannaninclude
EIAs [16, 25–27], RIAs [28, 29], and latex agglutination tests
[30, 31]. Only the latex agglutination test (Pastorex Aspergillus;
BioRad) is commercially available and has a higher detection
limit than the Platelia ELISA (15 ng/mL vs. 1 ng/mL).
Because galactomannan is a water-soluble carbohydrate, it
can be detected in samples of other fluids obtained from pa-
tients with invasive aspergillosis, including urine, CSF, pleural
fluid, and bronchoalveolar lavage (BAL) fluid [30, 32–34]. Al-
though the Platelia ELISA is not validated for detection of
galactomannan in these fluids, there is an increased tendency
to use samples of these fluids, in addition to serum, for di-
agnosis of invasive aspergillosis. In addition, galactomannan
can be detected in tissue specimens [35, 36]. The detection of
galactomannan in specimens other than serum specimens may
provide additional evidence for invasive aspergillosis via a non-
invasive method and may help to exclude false-positiveorfalse-
negative test results obtained using serum samples.
The literature was systematically reviewed to describe the
utility of galactomannan antigen detection in specimens other
than serum for the diagnosis of invasive aspergillosis. PubMed
(National Center for Biotechnology Information, National In-
stitutes of Health) was used to search for English-language
articles published since 1966 with use of the terms “galacto-
mannan detection,” “antigen,” “Aspergillus,” “invasive asper-
gillosis,” “diagnosis,” “ELISA,” “latex agglutination,”“culture,”
“tissue,” “urine,” “bronchoalveolar lavage fluid,” and “cere-
GALACTOMANNAN DETECTION IN
Because urine specimen collection does not require invasive
procedures, it appearstobealogicalspecimentouseforantigen
detection. Larger volumes could beexaminedandexaminations
could be more frequent, thereby improving the sensitivity of
the ELISA. Galactomannan can be detected in the urine spec-
imens of patients with invasive pulmonary aspergillosis, indi-
cating that at least a fraction of the circulating galactomannan
is cleared renally [16, 26, 30, 37, 38]. When galactomannan is
concentrated in the urine, testing of urine samples would yield
positive results for patients with low levels of circulating gal-
actomannan and therefore would be an early indicator of
However, little is known about the pharmacokinetics of gal-
actomannan and clearance by the kidney. When urine blots for
neutropenic patients were probed with the anti-galactomannan
EB-A1 antibody, diffuse staining was seen at molecular masses
of 145 kDa, and a weak band was seen at 21 kDa . In
addition, the molecular mass of galactomannan in the urine of
rabbits with invasive aspergillosis was also 18–21 kDa [37, 39].
The galactomannan-containing antigen in serum, as described
by Lehmann and Reiss , had a mass of 1125 kDa. It was
suggested that an increased size resulted from complexing of
the polysaccharide to protein in vivo . Within 24 h after
intravenous injection, 35% of galactomannan was excretedinto
the urine of immunocompetent rabbits . The detection
thresholds in serum and urine samples have been reported to
be about the same: 1 ng/mL [16, 42, 43]. Several authors have
suggested that urine is a better specimen for galactomannan
detection than is serum [26, 30, 37]. With use of EIA and RIA,
galactomannan could be found in urine from 7 of 13 patients
with invasive aspergillosis but in only 2 serum samples from
12 patients with invasive aspergillosis . Ansorg et al. 
used the latex agglutination test, and the sensitivity was in-
creased by concentrating the urine and lengtheningthereaction
time to up to 10 min. This yielded a sensitivity, specificity,
positive predictive value, and negative predictive value of an-
tigenuria for autopsy-proven aspergillosis and clinically sus-
pected Aspergillus infection of 57%, 53%, 31%, and 77%, re-
spectively . With use of the same monoclonal antibody as
than serum samples for galactomannan detection in one pro-
spective study . In contrast, the superiority of serum in
comparison to urine has been described in other reports [16,
Galactomannan in Nonserum Specimens • CID 2004:39 (15 November) • 1469
for the detection of galactomannan in serum, urine, and BAL
samples collected from 105 patients with hematologicdisorders
who received empirical amphotericin B to treat suspected fun-
gal infection. Antigen was detected in urine from only 2 of 5
patients with proven aspergillosis. Concentration of the urine
10-fold yielded only 1 additional urine sample with a galac-
tomannan-positive result. Stynen et al.  reported a sensi-
tivity of only 71% when urine samples were used, whereas
serum samples had a sensitivity of 100%. However, the value
of antigen detection in urine by one of the commercially avail-
able tests remains unclear because of the limited number of
patients with proven invasive aspergillosis and the predomi-
nantly retrospective study design of most published studies.
Differences in test results could also be explained by the dif-
ferent pretreatment procedures used for the urine samples (ta-
An important drawback of testing urine specimens is the
occurrence of false-positive results, which varies between 8%
and 47% [16, 30, 44]. The Platelia ELISA resulted in fewer
false-positive results (8%) than did the latex agglutination test
(42%) [16, 44]. Several explanations for false-positive results
have been suggested. Airborne fungi contaminating the urine
may cross-react with the EB-A2 monoclonal antibody .
Urinary antigen may reflect growth of a contaminating Asper-
gillus strain in urine or result from Aspergillus infection of the
kidney . Finally, the use of immunosuppressive agents, es-
pecially cyclophosphamide, has been associated with false reac-
tivity in urine specimens from rats . Specificity may be
improved by the examination of at least 2 consecutive urine
samples, by use of a higher cutoff, and by applying a longer
reaction time instead of concentrating the urine [27, 30, 47].
False-negative urine reactivity has also been reported and
may be associated with false-negative serum reactivity [37, 39,
48]. Alternatively, the presence of antibodies against Aspergillus
that bind to circulating antigen may cause smaller amounts of
galactomannan to be cleared by the kidney, resulting in lower
Little is known regarding the correlation between galacto-
mannan detection in urine and disease progression. Antige-
nemia preceded antigenuria in all patients (
or suspected invasive aspergillosis, as determinedbythePlatelia
ELISA . In that study, galactomannan was detected inurine
samples obtained from 4 patients shortly before death. Urine
samples from 2 other patients yielded positive results on the
day that these patients died. In contrast, when the less sensitive
latex agglutination test was used, antigenuria preceded anti-
genemia in 9 of 12 patients either with proven invasive asper-
gillosis or who were serologically suspected of exposure to As-
pergillus species . In an animal model, increasing urine
galactomannan concentrations corresponded with disease pro-
gression . Urinary galactomannan levels roughly paralleled
) with proven
n p 7
the extent of disease in one prospective study that used the
latex agglutination test . In that study, only 1 of 5 patients
who were apparently cured of their aspergillosis had galacto-
mannan detected in urine samples, as opposed to 6 of 8patients
who died . This might suggest a correlation between the
presence of antigenuria and fungal burden.
GALACTOMANNAN DETECTION IN
BAL FLUID SPECIMENS
The lungs are affected in most cases of invasive aspergillosis,
reflecting the primary site of entry of Aspergillus conidia. Be-
cause galactomannan is predominantly released by Aspergillus
hyphae during growth and to a much lesser extent by conidia,
detection of galactomannan in BAL fluid provides better evi-
dence for Aspergillus infection than do culture [49, 50] or PCRs
that do not discriminate between contaminating conidia and
hyphae [51–53]. However, BAL samples obtained from patients
colonized with Aspergillus, such as patients withlate-stageAIDS
and lung transplant recipients, show ELISA reactivity in the
absence of invasive disease. Several cutoff values have been
described for galactomannan detection by the Platelia ELISA
in BAL fluid, ranging from 0.17 to 1 ng/mL [42, 43].
Galactomannan could be detected by the latex agglutination
test in 43% of 30 BAL samples obtained from 42 patients with
pulmonary aspergillosis, of whom 38 had invasive pulmonary
aspergillosis . By use of the Platelia ELISA, galactomannan
was detected in BAL fluid from 2 of 3  and from 7 of 10
 patients with proven invasive aspergillosis. In the former
study, cultures for fungi yielded negative results for all BAL
samples that tested positive for galactomannan, although mi-
croscopic examination showed hyphae in 2 different samples
from a single patient .
Some investigators have differentiated between BAL and
bronchial lavage [8, 54, 56]. Evidence of Aspergillusinbronchial
lavage fluid could be demonstrated more frequently by antigen
detection than by culture. It was hypothesized that in the bron-
chi there may be more Aspergillus hyphae, with subsequently
more galactomannan release, than in the alveoli [49, 54]. In
contrast, for BAL fluid, culture more often yielded positive
results than did antigen detection. Possible reasons for this
finding are that very small conidia are predominantly found
in the alveoli or that the released galactomannan is diluted by
the lavage to a level below the lower detection threshold of the
In contrast to culture of BAL fluid, antigen detection in BAL
specimens, when combined with high-resolutionCT,couldlead
to earlier diagnosis and, subsequently, earlier treatment, re-
sulting in better survival rates [58, 59]. A prospective study
involving patients with hematologic disorders who hadinvasive
pulmonary aspergillosis reported positive predictive and neg-
ative predictive values of 100% when galactomannan detection
Table 1. Galactomannan detection in urine samples obtained from patients with invasive aspergillosis.
patientsStudy design Underlying condition(s)
of the urine
No. of patients
No. of patients
who were cured/
no. who died
Dupont et al., 1987 
Proven13 NR Hematologic malignancies, other hema-
tologic diseases, breast cancer,
inflammatory diseases, BMT
Hematologic malignancy, aplastic
Hematologic malignancy, BMT
Suspected8 NR DialysisRIA/ELISA0/0a
Haynes et al., 1990 :
proven or probable
Rogers et al., 1990 :
3 Prospective Filtration Immunoblot2 NR
8 Prospective Hematologic malignancy, inborn errors
of metabolism, solid tumor, aplastic
anemia, congenital immunodeficiency
Dialysis Inhibition ELISA44%NR
Ansorg et al., 1994 
Stynen et al., 1995 
Salonen et al., 2000 
Hematologic malignancy, BMT
Hematologic malignancy, BMT
Hematologic malignancy, BMT
Hematologic malignancy, aplastic anemia
Hematologic malignancy, aplastic anemia
Hematologic malignancy, BMT
Hematologic malignancy, SCT
aRIA result/ELISA result.
bUnconcentrated urine/concentrated urine sample.
cClassification according to the European Organization for Research and Treatment of Cancer Mycoses Study Group consensus definitions .
BMT, bone marrow transplantation; CGD, chronic granulomatous disease; NR, not reported; Pastorex, latex agglutination test; Platelia, sandwich ELISA; SCT, stem cell transplantation
Galactomannan in Nonserum Specimens • CID 2004:39 (15 November) • 1471
Table 2. Galactomannan detection in CSF samples obtained from patients with CNS aspergillosis.
patients Study designUnderlying condition
No. of patients
Kami et al., 1999 Proven5 NRHematologic malignancyPastorex and
Verweij et al., 1999 
Machetti et al., 2000 
Nenoff et al., 2001 
Viscoli et al., 2002 
Moling et al., 2002 
Diabetes mellitus type II
Chronic alcohol abuse
reported; Pastorex, latex agglutination test; Platelia, sandwich ELISA.
BMT, bone marrow transplantation; EORTC-MSG, European Organization for Research and Treatment of Cancer Mycoses Study Group; NR, not
was combined with systematic high-resolution CT . How-
ever, among the patients categorized as having invasive pul-
monary aspergillosis ( ), only 2 patients had proven in-n p 17
vasive pulmonary aspergillosis, whereas the others were
categorized as having probable (
invasive pulmonary aspergillosis. (In that study, a fourth group
of patients was defined in addition to the classification of the
European Organization for Research and Treatment of Cancer
Mycoses Study Group . Thiscategoryofsuspectedinfection
had a higher probability of infection than did those classified
as having possible infection but lower than did those classified
as having probable infection.) Sensitivity of galactomannande-
tection in BAL fluid was higher with the Platelia ELISA (85%–
100%)  than with the latex agglutination test (73%–83%)
[58, 60], whereas the sensitivity of antigen detection in serum
was much lower for both tests (47% and 41%–45% for the
Platelia ELISA and the latex agglutination test, respectively).
Combination of PCR and antigen detection could result in
higher sensitivity and specificity, because each method refers
to different fungal targets.
) or suspected ()n p 11n p 4
GALACTOMANNAN DETECTION IN
The CNS is the most common secondary site of invasive as-
pergillosis and is affected in 10%–20% of all cases of invasive
aspergillosis [4, 32]. The prognosis for invasive aspergillosis of
the CNS is extremely poor in immunocompromised patients.
Diagnosis of CNS aspergillosis is very difficult, and even brain
biopsies do not always result in a clear diagnosis . Culture
of CSF seldom yields positiveresults[4,62],andbothchemistry
findings in CSF and CT results are often not specific [63–65].
Galactomannan was detected in CSF specimens obtained
from patients with CNS aspergillosis caused by A. fumigatus,
Aspergillus terreus, and Aspergillus flavus by use of the Platelia
ELISA, RIA , and Western blotting, respectively [63, 67].
The cutoff for CSF samples is probably lower than that for
serum samples , which may be explained by a lower back-
ground reactivity of CSF. The CSF galactomannan indices of
patients with probable CNS aspergillosis were statistically sig-
nificant higher than were those of control patients with other
neurological diseases . The median CSF galactomannan
index was 10.52 in patients, compared with 0.287 in control
subjects . A summary of reports that describe the use of
the Platelia ELISA for the detection of galactomannan in CSF
is shown in table 2. The sensitivity and specificity were 80%
and 100%, respectively, in a study describing 5 patients with
proven CNS aspergillosis . Culture of CSF samples yielded
negative results, but PCR yielded positive resultsforallpatients.
False-negative ELISA reactivity of CSF was reported in apatient
with documented Aspergillus meningitis, although A. fumigatus
was cultured from the CSF and PCR yielded positive results
. In contrast, others report galactomannan detection to be
more successful than PCR .
There is evidence that galactomannan detection in CSF by
the Platelia ELISA may result in earlier diagnosis of CNS as-
pergillosis [63, 70]. In 1 patient, galactomannan could be de-
tected in the CSF sample 45 days before culture results were
positive . In another patient with Aspergillus meningitis,
culture of CSF yielded positive results . In addition to early
diagnosis of the infection, the course of the antigen titer has
been described to correspond with theclinicalresponsetotreat-
ment [63, 68].
GALACTOMANNAN DETECTION IN
Although the demonstration of Aspergillus in tissue is the ref-
erence standard for diagnosis of invasive aspergillosis, a defin-
itive diagnosis is possible only after identification of the fungus
cultured from that tissue, because the microscopic morphology
of many other hyalohyphomycetes, such as Scedosporium spe-
cies and Fusarium species, is often indistinguishable from that
1472 • CID 2004:39 (15 November) • Klont et al.
of Aspergillus species. However, up to 70% of tissues showing
septate hyphae yielded negative results by culture, thereby pre-
venting a definitive diagnosis . Other techniques, such as
PCR, in situ hybridization, and immunohistochemical tech-
niques, may prove to be useful tools when culture results are
negative and rapid identification to species level is necessary
. Immunohistochemical tissueanalyseshavebeendescribed
until now with 2 commercially availablemonoclonalantibodies
against galactomannan (EB-A1 and Mab-WF-AF-1), as well as
with the latex agglutination test and Platelia ELISA [35, 72–
74]. A protocol concerning the processing of solid-tissue spec-
imens for the Platelia test has been described on the Internet
. The monoclonal antibody EB-A1 showed good perfor-
mance in the detection of Aspergillus hyphae in routinely pro-
cessed tissue sections, providing generic identification .
This method was not superior to histological examinationwith
conventional stains for demonstrating the presence of an in-
fection . Another report described the use of Mab-WF-AF-
1 (DAKO) on paraffin-embedded sections obtained from pe-
diatric patients with invasive aspergillosis . The antibody
was highly immunoreactive with cell wall, septa, and cytoplasm
of Aspergillus species. This antibody is specific for A. fumigatus,
A. flavus, and Aspergillus niger. In necrotic tissue specimens,
immunohistochemical techniques may perform better than in
situ hybridization. Degradation of nucleic acid could affect the
performance of in situ hybridization, whereas this is less likely
to affect immunohistochemical analysis because Aspergillus
remnants in the cytoplasm of some phagocytic cells can be
detected by thismethod [72, 76]. Finally,acasereportdescribed
the use of the latex agglutination test in detecting galactoman-
nan in a brain biopsy specimen . This tissue was first
grounded in sterile 0.9% NaCl and centrifuged, and finally the
supernatant was used for galactomannan detection. The latex
agglutination test yielded positive results, but a fungal infection
could not be confirmed with microscopic evaluation and cul-
ture of the tissue. A cotton swab used to obtain the brain
specimen was found to have caused the reactivity. Because
wooden toothpicks also have been shown to cause reactivity
, cellulose was hypothesized to be responsible for the false-
GALACTOMANNAN DETECTION IN
By use of an inhibition ELISA, galactomannan antigen levels
exceeding 50 ng/mL were documented in cyst fluids from 2
patients with polycystic kidney disease . Although culture
of samples of these fluids was not done, culture of kidney
epithelium yielded Paecilomyces and Penicillium species. It is
possible that these fungi may have cross-reacted with the in-
hibition ELISA. Galactomannan could also be detected in a
subphrenic abscess of a 4-year-old boy with chronic granulo-
matous disease who had invasive aspergillosis . High levels
of galactomannan (70 and 48 ng/mL) were detected in 2 fine-
needle aspirations of the subphrenic abscess, and culture of
both specimens yielded A. fumigatus. Neither circulating As-
pergillus DNA nor galactomannan were detected, suggesting
that encapsulation of the abscess prevented leakageof surrogate
markers to the serum. Finally, galactomannan was detected in
pus specimens from patients with fungal rhinosinusitis .
Review of the literature indicates that, at best, antigendetection
in urine, BAL fluid, and CSF specimens is a promising diag-
nostic tool in addition to serum monitoring. The evidencethus
far is based on case reports, predominantly retrospective stud-
ies, often heterogeneous patient populations, various assays,
and limited numbers of cases of proven infection. Clearly,well-
designed prospective studies with systematic sampling that
make use of consensus case definitions are needed to compare
the performance of antigen detection in samples other than
serum samples with that in serum samples. These will also
temporal relationships with serum. In addition, more insight
into the kinetics of galactomannan is needed to understandthe
release and clearance of galactomannan during infection in
different patient groups. When these conditions are met, op-
timal, cost-effective strategies can be designed that incorporate
analysis of various samples and high-resolution CT.
Potential conflicts of interest.
All authors: No conflict.
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