Eur J Pediatr (2002) 161: 563–574
DOI 10.1007/s00431-002-1041-6
R EV IE W
Frank-Michael C. Müller Æ Andreas Trusen
Michael Weig
Clinical manifestations and diagnosis of invasive aspergillosis
in immunocompromised children
Received: 14 December 2001 / Revised: 1 June 2002 and 11 July 2002 / Accepted: 12 July 2002 / Published online: 11 September 2002

Springer-Verlag 2002
Abstract Invasive aspergillosis (IA) is a serious life-
threatening complication in immunocompromised
children. The commonest risk groups are children with
acquired immunodeficiency syndrome, leukaemia, cor-
ticosteroid and other immunosuppressive therapy,
chronic granulomatous disease and severe combined
immunodeficiency as well as neonates. The clinical
manifestations are heterogeneous and many organ sys-
tems can be involved. Diagnosis based on the clinical
presentation alone is cumbersome. Innovative and sen-
sitive laboratory test systems which detect fungal anti-
gens or DNA in clinical specimens have been recently
developed. Specific Aspergillus antibody detection using
recombinant antigen technique has also been intro-
duced. Although each individual technique has draw-
backs, the combined use of culture with antigen and
antibody ELISA as well as PCR should result in an
earlier and more definitive diagnosis of IA in children
presenting with clinical and/or radiological signs of
aspergillosis. In high risk children these methods are
valuable for serial screening and early detection of
Aspergillus infection. The implementation of accurate
diagnostic criteria and standardised diagnostic flow
charts in children at risk will lead to a better outcome of
IA in the future. Conclusion: definite, well-timed early
F-M.C. Müller
Department of Paediatrics and Institute for Molecular Biology
of Infection, University of Würzburg, Würzburg, Germany
A. Trusen
Department of Paediatric Radiology,
Institute for Diagnostic Radiology,
University of Würzburg, Würzburg, Germany
M. Weig
Institute for Bacteriology,
University of Göttingen, Göttingen, Germany
F-M. C. Müller (&)
Present address: Department of Paediatrics,
Haematology and Oncology, University of Heidelberg,
Im Neuenheimer Feld 150, 69120 Heidelberg, Germany,
Tel.: +49-6221-564555, Fax: +49-6221-564559,
e-mail: Frank-Michael_Mueller@med.uni-heidelberg.de
diagnosis and sufficient therapy is elementary for a
successful outcome of invasive aspergillosis in immu-
nocompromised children. To date, the diagnosis of in-
vasive aspergillosis remains a combination of clinical
presentation, radiology and microbiological tests.
Keywords Antibody Æ Antigen Æ Immunocompromised
children Æ Invasive aspergillosis Æ Molecular diagnosis
Abbreviations BAL bronchoalveolar lavage Æ CGD
chronic granulomatous disease Æ GM galactomannan Æ
IA invasive aspergillosis Æ SCID severe combine
immunodeficiency syndrome
Introduction
Invasive aspergillosis (IA) is an important life threat-
ening infectious complication in immunocompromised
children suffering from cancer disease, inherited and
acquired immunodeficiencies as well as in neonates [1,
27, 51, 65, 75, 94, 96,97]. Immunocompromised children
are at risk to acquire Aspergillus in the community or as
a nosocomial infection in the hospital environment.
Hospital construction work, renovation and contami-
nated air conditioning systems are well known sources
for nosocomial aspergillosis [25, 96,97]. The most rele-
vant predisposing factors to the development of IA in
children are granulocytopenia, corticosteroid and other
immunosuppressive therapies, organ transplantation
(especially heart-lung transplantation), quantitative and
qualitative immunodeficiencies, such as chronic granul-
omatous disease (CGD) and severe combined immuno-
deficiency (SCID) [97]. Children receiving high doses of
corticosteroid therapy including those with auto-im-
mune disease, organ transplants, lymphoma, brain
tumours, and allogeneic stem cell as well as bone mar-
row transplants (particularly with graft-versus-host dis-
ease) are at particular risk. Cushing syndrome with
endogenous hypercortisolaemia is another risk factor
for development of IA. Patients receiving high dose
564
cytotoxic chemotherapy for leukaemia, lymphoma, and
bone marrow transplantation which results in profound
and persistent granulocytopenia are at high risk for de-
velopment of invasive disseminated aspergillosis as a
complication of pulmonary infection [97].
While the diagnosis of cutaneous aspergillosis can
usually be made during life, the diagnosis of IA is in the
majority of cases made at autopsy [26,55]. Early diag-
nosis of IA in the immunocompromised child as well as
microbiological therapy monitoring is still cumbersome
as immunocompromised children rarely display typical
inflammatory responses. The interpretation of radio-
graphs is difficult especially during the early stage of the
disease. In many cases, invasive diagnostic procedures
cannot be performed because of the severity of the un-
derlying disease. Empirically, antifungal therapy is often
initiated before a definitive diagnosis is obtained. Due to
impaired lymphocyte function due to the underlying
disease and/or chemotherapy, antibody production is
usually low or completely absent, but may be useful to
validate cases retrospectively [97]. DNA amplification
methods have been developed, but the interpretation
of positive results, especially from bronchoalveolar
lavage (BAL) is still controversial. In children at risk,
Aspergillus-associated diseases can affect numerous
body sites and organs (Table 1). The clinical manifes-
tations of invasive pulmonary aspergillosis are multi-
faceted (Table 2). This article summarises clinical
manifestations of and the current diagnostic approaches
to IA in immunocompromised children.
Clinical manifestations of invasive aspergillosis
in immunocompromised children
Human immunodeficiency virus infection
Invasive pulmonary aspergillosis is by far the most
common presentation of aspergillosis in children with
AIDS [20, 45, 96,97]. IA was diagnosed in 7 (1.5%) of
Table 1. Clinical presentation of aspergillosis in children
Localised:
Otomycosis
Ceratitis
Cutaneous aspergillosis
Allergic:
Extrinsic allergic alveolitis
Extrinsic asthma
Allergic bronchopulmonary aspergillosis
Allergic Aspergillus sinusitis
Saprophytic:
Pulmonary aspergilloma
Invasive:
Bronchopneumonia
Necrotising tracheobronchitis
Invasive sinusitis
Chronic necrotising aspergillosis
Local extension to intrathoracic structures
Disseminated aspergillosis
Table 2. Clinical features of invasive pulmonary infection caused
by Aspergillus spp. in immunocompromised children
Leading symptoms:
Persistent or recurrent fever in persistently granulocytopenic
patients
Nonproductive cough
Occasional adventitious breath sounds, pleuritic pain,
pleural rub and haemoptysis
Clinical manifestations:
Bronchopneumonia
Segment or lobar consolidation
Cavity formation
Pleural effusion
Pulmonary vascular invasion, thrombosis, and infarction
Dissemination to extrapulmonary tissues
Invasion of chest wall, diaphragm, pericardium, and myocardium
Involvement of trachea to cause airway obstruction
Acute pancoast’s syndrome
Fistulas:
Bronchoarterial
Bronchopleural
Bronchocutaneous
Chronic necrotising infection
Necrotising tracheobronchitis
473 children with HIV infection followed at the Pedi-
atric Branch of the National Cancer Institute from
1987 to 1995 [77]. Prolonged survival because of highly
active antiretroviral therapy, hospitalisation, a past
history of pulmonary or sinus infection(s), tissue trau-
ma, corticosteroid use, neutropenia and HIV related
impairment of the phagocytic activity of macrophages
and, neutrophils against Aspergillus spp. may all con-
tribute to the susceptibility of HIV-infected patients to
aspergillosis [25, 46, 65, 73,74]. Interestingly, some of
these HIV-infected children did not have granulocy-
topenia or concomitant corticosteroid therapy, sug-
gesting an intrinsic impairment in fungicidal activity
[97]. Less common manifestations include infections of
the bronchial tree, which may present as necrotising
tracheobronchitis, pseudomembranous tracheobronchi-
tis or obstructing aspergillosis, infections of the para-
nasal sinus, middle ear and mastoid, CNS, the skin and
various other sites. Disseminated disease may also oc-
cur [51,87].
Clinical signs and symptoms are often unspecific and
misleading. Presenting features in pulmonary aspergil-
losis may include fever, cough, chest pain, and respira-
tory distress. Acute respiratory distress and wheezing or
the production of fungal casts can occur with necrotising
(tracheo)bronchitis, and stridor with laryngotracheitis
[97].
Haematological malignancies
Despite aggressive medical and surgical treatment strat-
egies for aspergillosis, the overall mortality of IA in
children with haematological malignancies is currently
about 85% over the year after diagnosis [1]. Pulmonary
aspergillosis in immunocompromised children suffering

from cancer disease has several manifestations, including
pneumonia, haemoptysis, and invasion of contiguous
intrathoracic structures. A common finding of invasive
pulmonary aspergillosis is prolonged or recurrent fever in
a persistently granulocytopenic or corticosteroid-treated
patient with pulmonary infiltrates [97,98]. Development
of pulmonary infiltrates may be initially absent due to the
weak inflammatory response. Non-productive cough,
pleuritic pain, and later haemoptysis are present in the
course of invasive pulmonary aspergillosis, reflecting
the potential of Aspergillus to invade blood vessels.
A persistently febrile granulocytopenic patient with
haematological malignancy, pulmonary infiltrate, and
haemoptysis has a high probability of having IA.
Haemoptysis is a leading symptom of invasive pulmonary
aspergillosis, particularly in granulocytopenic patients.
Two patterns of pulmonary haemorrhage and haemopt-
ysis may develop in granulocytopenic patients: The first is
that of haemorrhagic infarction due to vascular invasion.
The second pattern is the formation of mycotic aneu-
rysms during recovery from granulocytopenia and rup-
ture can cause potentially fatal haemoptysis [97].
Invasive pulmonary aspergillosis is not constrained
by anatomical barriers to the parenchyma of the lung.
Aspergillus spp. may invade through the visceral pleura
to the pleural space, intercostal muscles, ribs, parietal
pericardium, or great vessels. Once within the pericardial
space, hyphal elements may cause a pericardial effusion
and may continue to extend into the epicardium and
myocardium, causing myocardial infarction [7,97]. The
pericardial effusion may accumulate rapidly, leading to
pericardial tamponade. Invasion of the ribs and inter-
costal nerves may cause severe pain.
The CNS is the most common target organ of hae-
matogenous disseminated aspergillosis in children.
Manifestations of CNS aspergillosis include focal sei-
zures, hemiparesis, and cranial nerve palsies [97]. Dis-
seminated aspergillosis may also involve the eye, skin,
liver, gastrointestinal tract, kidneys, bones, and the
thyroid. Primary cutaneous aspergillosis has been asso-
ciated with Hickman intravenous catheters [3,95].
Aspergillus cutaneous lesions of haematogenous origin
in granulocytopenic children tend to appear on the ex-
tremities and may initially appear as purpuric nodules.
Corticosteroid and other immunosuppressive therapies
Corticosteroids directly increase the growth rate of
Aspergillus fumigatus and Aspergillus flavus. In addition,
corticosteroids reduce pulmonary macrophage killing of
Aspergillus conidia and neutrophil killing of Aspergillus
hyphae [88]. Patients receiving high doses of corticos-
teroid therapy are at increased risk for the development
of invasive aspergillosis: patients suffering from lym-
phoma, brain tumours, auto-immune diseases, organ
transplants, and bone marrow transplants (particularly
with graft-versus-host disease) [97]. Steroid-treated
allogeneic bone marrow transplant recipients are at
565
particularly high risk. Cushing syndrome, an endoge-
nous hypercortisolaemia, also predisposes to the devel-
opment of IA [97].
Solid organ transplantation
IA following solid organ transplantation remains a
major cause of morbiditiy and mortality. Acute rejection
after solid organ transplantation may not be an inde-
pendent variable as it reflects additional immunosup-
pression [88]. Rates of Aspergillus infection are
particularly high in lung transplant recipients (17%–
26%), followed by heart transplantation (2%–13%),
and renal transplantation (0.5%–10%). In lung trans-
plant patients, colonisation of the tracheobronchial tree
prior to transplantation, especially in cystic fibrosis
patients, does not increase the risk for IA [88]. In
corticosteroid-treated patients, including transplant
recipients, fever is often absent. Chest pain is common
and usually mild and non-specific but it may be pleuritic.
Inherited immune deficiency states
Chronic granulomatous disease
Patients with CGD have about a 33% lifetime risk of
IA. A. fumigatus is the most common cause of IA in
children with CGD. In a recent report on 368 patients
with CGD registered in the United States, pneumonia
was the most prevalent infection and Aspergillus was the
most prevalent cause. Sepsis and/or pneumonia due to
Aspergillus was the most common cause of death in this
group [102]. However, Aspergillus nidulans occurs with
an unusually high frequency in patients with CGD
compared with other groups of immunocompromised
hosts [97]. Aspergillus nidulans infection does not re-
spond to amphotericin B. IA may be the presenting
manifestation of CGD. In two long-term follow-up
studies of patients suffering from CGD, pneumonia and/
or sepsis due to Aspergillus spp. have been found the
major cause of complications and death [54,102]. Sen-
sitisation to Aspergillus may also occur [23]. Itraconaz-
ole prophylaxis is partially successful and voriconazole
may be an alternative in the future [64,99].
Severe combined immunodeficiency
IA occurs in about 4% of patients with SCID, usually in
the first 2 years of life [81]. It is almost invariably fatal,
with current approaches to diagnosis and treatment.
Term and preterm infants
Neonates are at increased risk for developing IA due to
their immature phagocytic capacity, corticosteroids, and
566
prolonged ventilation and hospitalisation [27, 70, 75,76].
In a large literature review over a period of 20 years on
aspergillosis during the first 3 months of life, primary
cutaneous and gastrointestinal aspergillosis were re-
ported almost exclusively in premature neonates, and
invasive pulmonary and disseminated aspergillosis as
well as aspergillosis of the CNS were mainly observed in
infants born at term [27,67] Prematurity, CGD, and a
complex of diarrhoea, dehydration, malnutrition, and
bacterial infections were identified as the main risk fac-
tors for developing IA [27]. Distinct clinical features or
specific radiographic signs are not present in cases of IA
that occur during the first 3 months of life. Even in cases
of widely disseminated disease, blood cultures were only
rarely positive, and cultures of CSF are usually negative
in infants with CNS involvement. Therefore the diag-
nosis is quite often established only after death [97]. IA
should be considered in very low birth weight infants
presenting with skin lesions, in the presence of a new
oral lesion, in the case of any acute vascular event in-
cluding pulmonary emboli or nodular and infarct-like
lung lesions, in the event of haemoptysis, and in the case
of persistent signs of infection despite antibiotic therapy
and negative cultures, especially when there is a combi-
nation of pulmonary and cerebral findings. The lungs
appear to be a frequent portal of entry [97]. Isolation of
Aspergillus spp. in any of these circumstances should be
regarded as strong indication for infection unless oth-
erwise excluded. Generally, any culture that is positive
for Aspergillus must be considered serious in the neo-
nate. Conversely, negative cultures do not exclude in-
fection and empirical therapy can be justified in certain
conditions.
Diagnosis of invasive aspergillosis
in immunocompromised children
Invasive pulmonary aspergillosis often develops in
immunocompromised children who are already
receiving broad-spectrum antibacterial therapy. Respi-
ratory distress and the appearance of new focal pul-
monary infiltrates in the setting of granulocytopenia is
suspicious for resistant bacteria, early cytomegalovirus
infection, and invasive fungal infections, including
Aspergillus spp. (especially A. fumigatus and A. flavus),
Trichosporon asahii, Fusarium spp., Scedosporium
apiospermum (Pseudallescheria boydii), and the Zygo-
mycetes (e.g. Rhizopus spp., Cunnighamella bertholet-
tiae).
Radiological findings in children
with invasive aspergillosis
There is a heterogeneous spectrum of radiological
findings in invasive pulmonary aspergillosis including
round pneumonic infiltrates, peripheral wedge-shaped
lesions and the typical ‘‘halo sign’’ around pneumonic
infiltrates, which can be detected by high resolution
CT [13, 14, 24, 36, 47, 61, 93, 97,103]. The air crescent
sign (also known as ‘‘Monod’s sign’’) is a late marker
that develops with the bone marrow recovery [14]. The
chest radiographic features of diffuse pulmonary infil-
trates in IA is distinct from those in adults [77]. Bi-
lateral involvement of the lungs and respiratory failure
are negative prognostic indicators of IA [19]. CT and
high resolution CT depict these lesions better and in
an earlier stage of disease compared to chest X-rays,
which is the routine imaging modality for patients with
clinical evidence of pneumonia. Standard CT has the
advantage of displaying the entire lungs, whereas ad-
ditional high resolution slices allow better delineation
of morphological changes and architecture of the
lungs. Because of the high sensitivity of the method,
CT in combination with high resolution slices of the
chest should be performed in patients at risk for pul-
monary IA presenting persistent fever under broad-
spectrum antibiotic therapy. This procedure is valuable
especially when the chest X-ray films show no abnor-
malities [53]. MRI offers some advantages as the ‘‘re-
verse-target sign’’ in the lung is highly suspicious for
necrotising pneumonia in invasive pulmonary asper-
gillosis [8,53]. Fig. 1 presents the case of a 14-year-old
boy suffering from a mediastinal lymphoma under
chemotherapy in aplasia (4 days neutrophil count 100/
ll): fever, cough, pleural pain and other specific
symptoms of IA were absent, the boy complained of
abdominal discomfort. A chest X-ray and MRI were
performed on the same day for staging purposes and
not for suspected IA. The chest X-ray film showed
discrete consolidations in the right lung (Fig. 1a). The
MRI was performed with frontal and transversal T1-
weighted fl2d-sequence with the breath-hold technique.
In the right upper and lower lobe the MRI demon-
strated new hyperintense round consolidations, par-
tially with central hypointense areas that are consistent
with aspergillosis (Fig. 1b and Fig. 1c). The CT scan
of the thorax performed after MRI showed the round
consolidations in the upper and lower lobe as well as
consolidation in the right perihilum (Fig. 1d and
Fig. 1e). This case illustrates the increased sensitivity
of CT scan and MRI over conventional chest X-ray
films in theearly stages of IA with the absence of fever
and clinical symptoms.
In CNS aspergillosis, CT scans with contrast
enhancement may initially reveal no focal abnormalities,
but at a later stage may demonstrate focal ring-enhancing
or haemorrhagic lesions. MRI scanning tends to identify
more lesions and may facilitate early detection of CNS
aspergillosis [8,53].
Biopsy and culture of clinical specimens
Definite diagnosis of IA depends on the cultivation of
Aspergillus spp. from sterile body sites. Likewise histo/
cytopathological demonstration of hyphal penetration
 567

Fig. 1. a A 14-year-old boy suffering from mediastinal lymphoma:

only discrete consolidations in the right lung are seen on a
conventional chest X-ray film. Fig. 1b MRI scan from the same day
in a frontal T1-weighted fl2d-sequence using the breath-hold
technique showing multiple hyperintense round consolidations,
partially with hypointense areas in the right upper and lower lobe
of the lung. Fig. 1c MRI scan from the same day in a transverse
T1-weighted fl2d-sequence using the breath-hold technique reveal-
ing a new hyperintense round consolidation in the right upper
lobe. Fig. 1d A transverse CT scan from the same day showing
round consolidations in the lower lobe. Fig. 1e A transverse CT
scan from the same day showing a perihilar consolidation on the
right side

in host tissue in combination with tissue damage and in debilitated children biopsy is often precluded because
situ hybridisation techniques or immunohistochemical of thrombocytopenia or other conditions that rule out
staining substantiate the diagnosis of IA. However, in invasive diagnostic procedures.
568
Respiratory tract specimens
The interpretation of results obtained from a culture of
respiratory tract specimens is difficult because of the
ubiquitous nature of airborne conidia and the risk of
accidental contamination. Although consensus has not
been reached, there are increasing data which show that
cultivation of Aspergillus from normally sterile sites (i.e.
open or percutaneous lung biopsy) and the presence of
Aspergillus in respiratory samples from immunocom-
promised children at risk for IA is highly indicative of
infection. The use of BAL has been studied by several
investigators and found to yield variable results with
sensitivities ranging from less than 25% to as much as
75% when analysed in tissue proven infection. Al-
though an A. fumigatus culture positive BAL fluid is
indicative of invasive disease in febrile granulocytope-
nic children with new pulmonary infiltrates, the absence
of hyphal elements or negative culture do not exclude
the diagnosis [39]. Aspergillus sinusitis may develop
before or simultaneously with invasive pulmonary as-
pergillosis. A biopsy of mucosal eschars may be per-
formed and culture of these ‘‘sentinel eschar’’ lesions
may reveal IA and prompt initiation of appropriate
therapy.
Lung biopsy
The lungs may be biopsied using a percutaneous tech-
nique, thoracoscopy or an open lung approach. A series
of 28 percutaneous biopsies in 24 children with sus-
pected IA showed that 10 were positive and 18 negative
[37]. Bleeding occurred in 13 instances and hastened one
death. Open lung biopsy is recommended when the di-
agnostic tests have not yielded a microbiological diag-
nosis in the setting of new infiltrates in a persistent
febrile granulocytopenic or otherwise immunosup-
pressed child. For patients with a localised infiltrate,
however, open lung biopsy will require a major thorac-
otomy using either a lateral or mediastinal approach.
Biopsy specimens should be obtained from the periph-
eral and central areas of abnormal lung tissue because
the distribution of Aspergillus hyphae may vary. Tho-
racoscopic biopsy for IA has not been reported in chil-
dren.
Blood culture methods
Although little explored, successful detection of IA
using blood culture methods are rarely reported in the
literature [21]. Considering the high frequency of IA
compared to the low ratio of fungaemia that are de-
tected using various blood culture systems, it seems
likely that a rapid clearance of fungal elements takes
place in the capillary beds of the host. The use of the
lysis-centrifugation system has been preferred by some
authors, but an optimal blood culture system for the
detection of filamentous fungi has so far not been
developed.
Serology
As clinical, radiological and culture diagnosis of IA is
difficult in paediatric patients A. fumigatus serology that
reliably leads to an early diagnosis could substantially
improve the clinical outcome [2, 6,12]. However, con-
ventional antigen and antibody tests are still hampered
by some major obstacles.
The usefulness of circulating galactomannan (GM),
an immunodominant polysaccharide – cell wall antigen
of Aspergillus spp. for the diagnosis of IA has been
studied extensively in the past [17, 19, 49, 50,80]. Re-
cently, the monoclonal antibody EB-A2 has successfully
been used in a direct double sandwich ELISA assay to
recognise the 1,5-b-D-galactofuranoside side chains of
circulating GM during IA (Platelia Aspergillus, Bio-
Rad) [17, 18, 71, 72,80]. This assay is very sensitive and
allows the detection of 0.5 to 1.0 ng GM/ml serum.
Numerous studies have documented the superior sensi-
tivity of this ELISA compared to the latex agglutination
test (Pastorex Aspergillus, Sanofi-Pasteur) [42, 48, 83,
91,101]. However, comprehensive data on GM detection
in paediatric patients at risk are lacking (Table 3). In a
well-performed prospective study to assess the value of
serial screening for circulating GM in 186 prolonged-
neutropenic and /or steroid-treated patients, Maertens
et al. [58] were able to demonstrate that the sensitivity of
serial GM monitoring (Platelia Aspergillus, BioRad) was
almost 93%. In more than 50% of cases, antigenaemia
was detected before clinical suspicion of IA. A drawback
of the sandwich GM antigen ELISA, however, is a high
frequency of false-positive results [83, 84,91]. It appears
that antigen detection methods have a low positive
predictive value, particularly in neutropenic patients
[41]. In accordance, a high rate of positive antigenaemia
in bone morrow transplant children without any sign of
infection has been observed [52], the reasons for which
are not fully understood. Different mechanisms have
been discussed such as transient presence of circulating
Aspergillus-GM antigen during neutropenia, cross-
reactivity of the test with antigens of other fungi and
soluble GM found in vegetable and cereal foods, intes-
tinal fungal colonisation or cross-reaction with uniden-
tified serum components, drugs or metabolites [84,85]. A
substantial problem in testing premature infants was
reported by Siemann et al. [78]. The group detected
83.3% false-positive test results (5/6) in infants with a
weight of 400 to 1320 g. The reasons remain speculative
and conclusive data from a larger population of pre-
mature infants have so far not been published.
A second problem in GM antigen testing is the fact
that true false-negative results have been documented in
IA patients [91,100]. Verweij et al. [92] published a de-
tailed record on the failure to detect circulating GM
antigen in serially obtained serum specimens and fungal
 569

Table 3. Serological studies in paediatric and adult patients suf- fibrosis; EORTC European Organisation for Research and Treat-
fering from Aspergillus-associated diseases reported in the litera- ment of Cancer; ICU intensive care unit; IPA invasive pulmonary
ture. (ABPA allergic bronchopulmonary aspergillosis; AO aspergillosis; LA latex agglutination; NPV negative predictive
aspergilloma; BMT bone marrow transplantation; CF cystic value; PPV positive predictive value)
Test Patients Aspergillus related Specimen Results Reference
disease examined

GM-LA Adult haematological 8 patients with proven Serum samples Sensitivity: 23% [41]
patients (n=88) IPA (69 serum samples), (n=872) Specificity: 98%
8 suspected cases PPV: 64%
(55 serum samples) False-positives: 11%
GM-LA and Adult patients 6 patients with proven Serum samples LA: [91]
GM-sandwich at risk for (42 serum samples) (n=532) Sensitivity: 70%
ELISA IA (n=61) and 4 with possible IA Specificity: 86%
(45 serum samples) ELISA:
Sensitivity: 90%
Specificity: 84%
GM-sandwich Paediatric and 9 patients with IA Serum samples GM detected in [83]
ELISA adult haematological (59 serum samples, (n=223) all Aspergillus
patients 5 proven cases, infected patients
2 paediatric patients) Serum more appro-
priate than urine
Detection limit:
0.5–1 ng GM/ml
False-positives: 8%
GM-sandwich Adult haematological 27 episodes of proven Serum samples Sensitivity: 92.6% [58]
ELISA patients (n=186) IA (276 serum samples) (n=2,172) Specificity: 95.4%
PPV: 93%
NPV: 95%
False-positives: 8%;
2 patients (5+9 serum
samples) remained
consistently negative
GM-sandwich Patients (n=247) with 6 infants without IA Serum samples False-positives: [78]
ELISA severe underlying diseases (10 serum samples) (n=783) 83.3% (5/6)
and 6 paediatric patients
with prolonged ICU stay
and a birth weight
of 400 – 1320 g
GM-sandwich Children aged 1 to 16 All children without Serum samples Every child had 1–4 [52]
ELISA years (n=7) clinical or radiological (n=47) false positive serum
signs of IA samples
Allogenic BMT children 19 false positive
(n=5) test results
Children with
haematological
malignancies (n=2)
GM-sandwich Children: aged 0 to 17 Children without IA Serum samples Sensitivity: 66.7% Letscher-Bru
ELISA years (n=48) (n=42) (n=223) (personal
BMT children (n=18) 6 children with IA Specificity: 47.6% communication)
Children suffering from (3 possible, 2 probable, PPV: 15.4%
cancer (n=30) 1 proven case, NPV: 96.7%
EORTC criteria) 22 children had false-
positive test results
(82 positive serum
samples)
GM-sandwich Paediatric (n=37) (mean Children (n=10) with Serum samples PPV: 83% [72]
ELISA age 8.5 years, range probable IA (164 serum (n=413)
5 months to 18 years) samples)
neutropenic and
BMT patients
13 A. fumigatus colonized False positive: 5.7%
CF paediatric patients Negative results in
all 13 colonised
CF children
G test 202 episodes in 179 patients 41 febrile episodes >321 plasma Sensitivity: 90% [66]
(determination (67% of underlying from proven samples
of 1,3-b-glucan) diseases were fungal infections
haematological disorders)
Patients were aged 59 febrile episodes from Specificity: 100%
2–99 years proven bacterial infections
570

Table 3. (Continued)
Test Patients Aspergillus related Specimen Results Reference
disease examined

102 febrile episodes PPV: 59%

from fever of unknown
origin
NPV: 97%
Recombinant Paediatric (n=27) and 23 CF patients (n=20) Serum samples IgE antibodies to [35]
Aspf4 and adult patients suffering suffering from ABPA (n=50) rAspf4 and rAspf6
Aspf6 IgE from CF (consisting of (15 paediatric patients) exclusively found
antibody- 20 Aspergillus sensitised in patients suffering
ELISA and 20 ABPA from ABPA
CF patients and 10 not Sensitivity: 100%
sensitised control Specificity: 100%
CF patients)
Recombinant Adult patients suffering Patients (n=13) with Serum samples AO: [100]
mitogillin various underlying proven AO (n=117)
antibody- diseases (n=36) (32 serum samples)
ELISA 20 patients with proven Sensitivity: 100%
IA (82 serum samples) Specificity: 95%
PPV: 95%
NPV: 100%
IA:
Sensitivity: 62%
Specificity: 95%
PPV: 93%
NPV: 72%

DNA in plasma samples of a non neutropenic 4-year-old
boy suffering from CGD who developed invasive pul-
monary aspergillosis. Absence of circulating antigen
in neutropenic patients with IA has been previously
reported [11,58]. False-negatives might result from
increased antibody titres that inhibit the detection of
circulating GM, a lessened GM release from the cell wall
of the fungus, strain variations in the efficiency to release
cell wall antigens, modification in the level of circulating
antigen by the immune response of the host, encapsu-
lation of the infection or low level blood vessels inva-
sion. To overcome this problem, serial screening of
patients at risk [58] and combined testing (circulating
antigen and specific antibody detection) seem valuable
[100].
Elevated plasma levels of 1,3-b-glucan, a fungal cell
wall constituent, can be determined with factor G, a
horseshoe-crab coagulation enzyme that is extremely
sensitive to this polysaccharide. In sera of rats with ex-
perimental IA, elevated 1,3-b-glucan levels paralleled the
development and progression the disease [28,63]. In a
study of 202 febrile episodes in patients aged 2–99 years,
the determination of plasma 1,3-b-glucan was found to
be a sensitive and specific test for invasive deep mycoses
and fungal febrile episodes (Table 3) [66].
So far, commercial antibody detection kits have
been based on preparations from conidial, mycelial,
cytoplasmatic, metabolic or cell-wall fractions of
A. fumigatus. These crude extracts contain complex and
undefined mixtures of antigenic polysaccharide, lipid
and protein components [31, 32, 33, 69]. Consequently,
problems arise such as cross-reaction of antigenic
determinants, lack of specificity and sensitivity, consid-
erable batch to batch variations, qualitative and quan-
titative differences in the composition of antigens and
contamination that occurs during the production pro-
cess. Aspergillus fumigatus is ubiquitous in nature and
antibodies to numerous Aspergillus antigens reflect a
pre-existing humoral situation rather than invasive dis-
ease. These disadvantages of conventional test systems
together with a reduced antibody response observed in
immunosuppressed children has led many paediatricians
to abstain from performing anti-Aspergillus antibody
detection. The diagnostic exploitation of selected im-
munodominant recombinant fungal antigens which can
be produced in large quantities could substantially im-
prove the serodiagnosis of aspergillosis and complement
the critical points of antigen detection. Recently the first
steps in identifying suitable antigens have been taken.
Using a cDNA encoding A. fumigatus allergens which
were cloned from a cDNA library displayed on a phage
surface, Hemmann et al. [15, 16,35] were able to identify
two antigens, an intracellular manganase superoxide
dismutase (rAsp f6) and a protein with unknown func-
tion (rAsp f4) which were recognised exclusively by IgE
from sera of CF patients with allergic bronchopulmo-
nary aspergillosis [35].
Weig et al. [100] were able to demonstrate that anti-
bodies to recombinant mitogillin can be used as a spe-
cific marker in the serodiagnosis of IA. This A. fumigatus
antigen is rare in resting conidia but is highly expressed
during invasive growth of the fungus [4,5]. Mitogillin is
one of the major A. fumigatus protein antigens and
elicits an early antibody response in the host. Anti-
mitogillin antibodies were detectable in GM-antigen
negative serum samples of patients suffering pulmonary
and disseminated IA, but were almost absent in speci-
mens of healthy controls.

Nucleic acid amplification methods
In the adult population, numerous PCR assays for the
diagnosis of aspergillosis utilising different DNA tar-
gets have been reported [22, 30, 40, 60, 62,89]. Various
DNA fragments within the 18S rRNA of Aspergillus
spp [22, 44, 59,104], a 747-bp fragment of genes
encoding alkaline protease from A. fumigatus for
detection in BAL fluid specimens [86] a 135-bp frag-
ment in the mDNA of Aspergillus spp., as well as a
310-bp [38] and a 401-bp fragment in the rDNA
complex of A. fumigatus have been amplified success-
fully from clinical samples of patients with IA. The
amplification of sequences from the IgE-binding site of
Aspergillus appears to lack sensitivity [68]. Generally,
PCR-based assays are very sensitive in the detection of
Aspergillus spp. in clinical specimens. However, since
Aspergillus spp. are ubiquitous in the environment and
known to colonise the respiratory tract without disease,
the interpretation of a positive amplification result
from respiratory specimens including BAL can be dif-
ficult [10,89]. PCR diagnosis is accurate in culture-
negative BAL fluid specimens from patients with
invasive pulmonary aspergillosis; however, it does not
differentiate between infection and colonisation [29]. Of
28 BAL samples from non immunosuppressed patients
without aspergillosis, 5 were PCR-positive in a study
by Tang et al. [86]. This problem is further reflected in
a study by Spreadbury et al. [79]: detection of the 26S
intergenic spacer region of the rDNA complex was
found positive with respiratory specimens from patients
with invasive disease, but positive results were also
noted with specimens from control patients. In a study
of early detection of Aspergillus infection after alloge-
neic stem cell transplantation by PCR, the sensitivity
and the negative predictive value were both 100%;
however, the positive predictive value of 44% was
rather low, but could be improved if calculations were
based on two positive PCR results [34]. Aspergillus
DNA was detected in serum by nested PCR in 31 of 33
patients with aspergilloma, in all four cases of IA, in
three of four patients with Aspergillus pyothorax, and
in one of four patients with allergic bronchopulmonary
aspergillosis, indicating a low rate of false-negative
PCR results [44]. These false-negative PCR results
could have been generated by inhibitory factors in the
samples [89,104]. Verweij et al. [89] suggested that the
combination of Aspergillus genus specific PCR and
sandwich GM-antigen ELISA might be beneficial for
use in the early diagnosis of IPA in patents with
haematological malignancies. Since false-positive and
false-negative GM-antigen results may be found by the
antigen-ELISA of serum samples, additional evidence
for the presence of invasive pulmonary aspergillosis
may provide detection of Aspergillus spp. in BAL fluid
by antigen-ELISA or PCR [89] and PCR of serum
samples [91]. For PCR, serum seems to be the most
suitable specimen to minimise false-positive results
since no organisms are usually isolated from these
571
samples [104]. For the diagnosis of aspergilloma from
serum samples, a nested PCR assay was found superior
to the latex agglutination test and GM-antigen detec-
tion by ELISA [43,104]. In one study by Hebart et al.
[34], prospective examination of blood samples by PCR
following allogeneic stem cell transplantation allowed
identification of patients at very high risk for IA.
The different PCR assays have been optimised with
special regard to minimise the risk of carryover con-
tamination, quantification of the fungal DNA load of a
clinical specimen, and to reduce the number of labo-
ratory steps and time to obtain the result by using the
TaqMan system and by the light cycler system [9,
56,57]. For the purposes of routine diagnostics of IA,
nucleic acid sequence-based amplification might offer
some advantages over PCR in the future, as results of
comparable sensitivity and specificity can be obtained
already within 6 h [57]. Only very limited data are
available comparing PCR in various body fluids (i.e.
blood versus or serum versus plasma). In prospective
trials of proven invasive aspergillosis, intra-/interlabo-
ratory reproducibility of the results, especially with
regard to standardised extraction procedures, must still
be verified [82]. Future trials will have to focus on the
value of nucleic acid amplification, antigen and anti-
body detection and metabolite screening methods in the
paediatric population. Specific conditions, such as
smaller blood volumes available for diagnostic proce-
dures have to be considered and diagnostic flow charts
should be standardised. In addition, the question
whether a PCR or antigen-ELISA-based pre-emptive
antifungal therapy will help to reduce Aspergillus-
related mortality in high risk children has to be further
elucidated.
Conclusion
New and promising diagnostic tools have been recently
developed and these will help to improve the conven-
tional diagnosis if IA, which was based mostly on the
occurrence of pulmonary infiltrates in patients at risk
suffering prolonged fever refractory to antibiotic therapy.
It can be expected that earlier and specific diagnosis of
IA will be associated with a better disease outcome in
immunocompromised children. In addition, these im-
provements will lead to a reduced number of children
receiving unnecessary empirical antifungal therapy
which has side-effects and the potential to develop drug
resistance.
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