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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tazobactam and correlations between in vitro, in vivo, and clin-

ical properties of the drug-antigen interaction. J Clin Microbiol

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8. Buchheidt D, Hummel M, Schleiermacher D, Spiess B,

Hehlmann R. Current molecular diagnostic approaches to sys-

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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: m.mennink@mmb.umcn.nl

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.