Invasive aspergillosis (IA) is the leading infection that causes
death in immunocompromised patients, including transplant
patients and those treated for haematological malignancy or
with high-dose corticosteroids. The incidence of IA varies
depending on the patient population, but is increasing in most
groups due to the more widespread use of immunosuppressive
therapy and more aggressive treatment regimens [1]. The mor-
tality rate from IA is as high as 50-90%, which is partly due to
the difficulty in establishing a diagnosis at the early stages of
infection, since presenting symptoms are often non-specific
and the sensitivity of cultures is low. Definitive proof of an
invasive fungal infection can only be obtained by showing inva-
sive growth in tissue [Figure 1] and culturing Aspergillus from
the same specimen. However, invasive procedures are often pre-
cluded due to severe thrombocytopenia and therefore non-
invasive tests and procedures have been evaluated in order to
establish a diagnosis with a high level of confidence.
Techniques to improve timely diagnosis have focused on the
detection of circulating surrogate markers released by the fun-
gus [2, 3]. With the development of non-culture based methods
such as PCR and antigen detection, circulating markers can be
detected early in patients with invasive disease [4]. Another
promising technique is high-resolution computed tomography
(HRCT).
Prevention
Prevention of infection appears to be the optimal strategy for
managing an infection that has a high mortality rate and is dif-
ficult to diagnose. However, the transmission of Aspergillus and
the pathogenesis of invasive infection remains poorly under-
stood. Patients are believed to become infected by inhalation of
Aspergillus spores (conidia) [Figure 2], which are present in the
ambient air. This is reflected by the fact that over 90% of
patients present with a pulmonary infection. Although many
hospital rooms for the care of high risk patients are equipped
with air filtration systems designed to reduce patient exposure
to airborne Aspergillus conidia, the incidence of infection is still
rising. There is increasing evidence that Aspergillus infection is
acquired outside the hospital and clinical disease then manifests
during hospitalisation when the patient is severely immuno-
suppressed. In addition, sources of infection other than air are
being investigated, following the observation that there can be
high levels of Aspergillus in hospital water and in showerheads.
This route of transmission might be of importance in certain
hospitals. Chemoprophylaxis with mould-active antifungal
drugs is another approach used to prevent IA, although a bene-
ficial effect on fungal infection-related mortality or overall mor-
tality has not been shown convincingly.
Surrogate markers:
galactofuranose(galf)-antigens
The availability of a commercial ELISA to detect Aspergillus
(Platelia Aspergillus (PA), BioRad, France) has resulted in a sig-
nificant improvement in the early diagnosis of IA. This assay,
now widely used throughout the world, uses the rat IgM mon-
oclonal antibody EB-A2, which binds the β(1-5)-galactofura-
nosyl (galf) side chains of the Aspergillus galactomannan (GM)
molecule and some other galf-containing antigens [Figure 3]
[3]. Circulating antigen may be detected at a mean of 8 days
(range, 1-27 days) before diagnosis can be made using alterna-
tive methods, and therefore prospective monitoring of serum is
a feasible approach in high risk patients. A major drawback is
the variability in performance of this assay (sensitivity 50 to
92.6%; specificity 94 to 99.6% in patients with haematological
malignancies). Although multiple factors are
believed to have an impact on the performance of
the assay [5], such as the cut-off used to define a
positive result, exposure of patients to mould-
active antifungal drugs was recently shown to
cause a significant reduction in sensitivity. In
addition, false positive ELISA reactivity was
observed in patients receiving certain β-lactam
antibiotics, including piperacillin-tazobactam or
amoxicillin-clavulanic acid. This reactivity is
probably due to cross-reacting galf-components
that originate from the mould Penicillium, which
is used for antibiotic production [6, 7].
Genomic fungal DNA
PCR detection of Aspergillus DNA from body
fluid samples using conserved or specific genome
sequences is a promising tool and is being used
increasingly as a diagnostic method [8]. In some studies, circu-
lating fungal DNA could be detected in blood at a median of 9
days before diagnosis by conventional methods. Furthermore,
quantitative PCR techniques, such as those that employ the
lightcycler or Taqman, can also be used for monitoring fungal
burden and a patient's response to antifungal therapy. There
are, however, a number of drawbacks associated with PCR
diagnosis. As with antigen detection, the performance of this
technique is affected when patients have been exposed to (pro-
phylactic) mould-active antifungal agents. There is no stan-
dardised PCR method for the detection of Aspergillus DNA,
which limits its broad use in clinical practice and precludes
comparisons between different diagnostic studies. The sensitiv-
ity of this technique was found to range from 57 to 100% in
F ungal Infections
Current diagnostic strategies for themanagement of invasive aspergillosis
As published in CLI February/March 2005
Invasive aspergillosis has become a leading cause of death amongst immunocompromised patients.
Because timely diagnosis of this fungal infection is essential for the survival of these patients, and invasive
procedures are often precluded, current diagnostic techniques are based on the detection of circulating
markers released by the fungus early in the disease course. This review discusses the advantages and lim-
itations of the current diagnostic strategies.
dis
ease
fo
cus
by Dr M. Mennink-Kersten and Dr P Verweij
Figure 1. Microscope image of a tissue biopsy showing sep-tate hyphae consistent with Aspergillus (Grocott stain).
Figure 2. Electron micrograph of a conidial head of Aspergillus.
patients with haematological malignancies, while the specifici-
ty ranged from 65 to 100%. In addition, the detection of fungal
DNA in consecutive blood samples is variable, indicating that a
high number of samples need to be collected in order to diag-
nose the majority of patients.
β-D-Glucan
A commercial method for the determination of 1,3-β-D-glucan
(BDG) has recently become available on the European market
(Fungitell, Associates of Cape Cod). This assay detects BDG,
which is a cell wall component of most medically important
fungi, including Aspergillus [9]. Furthermore, Aspergillus
secretes BDG into culture fluid during growth, and elevation of
the BDG level in plasma paralleled the development and extent
of Aspergillus infection in an animal model. A concentration of
80 pg/mL (cut-off value) of this polysaccharide can be detected
in the serum of IA patients by activation of factor G, a coagula-
tion factor of the horseshoe crab [9, 10]. A drawback of this
very sensitive test is that there are several factors that might lead
to a false positive diagnosis. Endotoxin-free and glucan-free
materials must be used. In addition, cellulose membrane-asso-
ciated haemodialysis, gauze sponges used during surgery and
patients treated with intravenous albumin and gamma globulin
could all give false positive results. The experience with this
assay has been limited with the exception of its use in Japan.
High-resolution computer
tomography
Characteristic lesions caused by invasive growing fungi can be
visualised using a high resolution CT scan (HRCT). These include
nodular lesions, wedge lesions, the halo sign and the air crescent
sign [Figure 4]. Nodular lesions and halo signs appear early in the
course of infection, while the air crescent sign is usually visible fol-
lowing recovery of the aplasia. Systematic HRCT combined with
aggressive surgical intervention in patients with single lesions was
shown to significantly improve survival compared with a strategy
based on HRCT in patients who presented with clinical symptoms.
Nevertheless, HRCT does not allow aetiological diagnosis and in
many patients with pulmonary fungal infection the lesions may be
atypical.
Management strategies
An empirical treatment strategy is commonly used for patients
with a persistent fever that does not respond to broad-spectrum
antibacterial therapy. Treatment with antifungal drugs is then
started promptly without mycological evidence of fungal infec-
tion. The availability of new potent antifungal drugs, and more
sensitive diagnostic tools, has created initiatives to evaluate new
management strategies. Intensive monitoring using surrogate
markers combined with HRCT might be a promising approach
in which the infection is detected early and patients who
require antifungal therapy are selected more accurately than
with the empirical strategy. Studies are now underway to eval-
uate the feasibility of this so called pre-emptive treatment strat-
egy. Practically, patients are monitored systematically, for exam-
ple, twice weekly, using surrogate markers during the period of
highest risk. Once antigen or fungal DNA is detected the patient
will undergo a HRCT in order to confirm the presence of an
invasive fungal infection and antifungal treatment will be initi-
ated. Alternatively, in patients with persistent fever but without
evidence of an invasive fungal infection (negative surrogate
markers) treatment may be withheld. Although this approach
remains to be evaluated for the management of invasive fungal
infections, similar approaches have been succesful with other
infectious diseases such as viral infections (CMV and EBV).
Conclusions
Although the diagnosis and management of IA remains very
difficult, new strategies are being evaluated that incorporate
surrogate markers. All the markers available to date have draw-
backs. However, when they are combined with other tests and
procedures, patients that require antifungal
therapy can be identified early in the course
of infection. Strategic studies are needed, not
only to understand the release and kinetics of
surrogate markers, but also to determine the
optimal sequence of events to enable early
diagnosis of infection.
References
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aspergillosis in 2002: an update. Eur J Clin
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aspergillosis. Lancet 2000; 355: 423-424.
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ical properties of the drug-antigen interaction. J Clin Microbiol
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8. Buchheidt D, Hummel M, Schleiermacher D, Spiess B,
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tological malignancies. Leuk Lymphoma 2004; 45: 463-468.
9. Obayashi T, Yoshida M, Mori T, et al. Plasma (1-->3)-beta-D-
glucan measurement in diagnosis of invasive deep mycosis and
fungal febrile episodes. Lancet 1995; 345: 17-20.
10. Odabasi Z, Mattiuzzi G, Estey E, et al. Beta-D-glucan as a
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The author
Monique A.S.H. Mennink-Kersten, Ph.D
Paul E. Verweij, MD, PhD
Department of Medical Microbiology and Nijmegen University
Center for Infectious Diseases,
Radboud University Nijmegen Medical Center,
The Netherlands
Correspondence to:
Monique A.S.H. Mennink-Kersten, Ph.D
Department of Medical Microbiology
Radboud University Nijmegen Medical Center
P.O. box 9101
6500 HB Nijmegen
The Netherlands
Tel.: +31 24 3613514
Fax: +31 24 3540216
Email: [email protected]
F ungal Infections As published in CLI February/March 2005
Figure 2. The Platelia Aspergillus ELISA technique. (A) The test uses microplatesthat are pre-coated with the rat monoclonal antibody EB-A2. The same antibody,coupled to peroxidase, is used as the detector. This is added to the wells, followedby the tested sample containing a galf-component. (B) After incubation for 90minutes at 37°C, plates are washed and TMB chromogen is added. After a fur-ther 30 minute incubation, the reaction is stopped with H2SO4 and the resultingyellow coloured product can be measured at 450 nm (C).
Figure 3. High-resolution CT scan showing a pulmonaryinfiltrate in a neutropenic patient. Although this patientwas diagnosed with an invasive pulmonary aspergillosis,the lesion lacks some typical characteristics suggestive ofinvasive fungal infection.
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