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Advances in diagnostic testing
P. E. VERWEIJ
Department of Medical Microbiology, University Medical Centre Nijmegen, and Nijmegen University Centre for InfectiousDiseases, Nijmegen, The Netherlands
Within the past decade surrogate markers have become important and reliable
tools for the early diagnosis of invasive aspergillosis. Surrogate markers include the
antigens galactomannan and (10/3)-b-D-glucan for which commercial assays are
available, as well as fungal DNA for which experimental PCR assays have been
developed. Although many clinical validation studies have been performed, the
kinetics of these markers are still largely unknown. Recent studies have addressed
several issues related to variables that interact with the performance of the assays,
including exposure to mould-active antifungal agents and the interpretive cut-off
levels. Insights gained as a result of these studies will help us to further optimize
management strategies and to determine the optimal test sequence.
Keywords Aspergillus PCR, diagnosis, galactomannan, (10/3)-b-D-glucan,
invasive aspergillosis
Introduction
Surrogate markers have become an important tool for
the management of invasive aspergillosis. Assays that
detect circulating markers are used as an early indicator
of opportunistic fungal disease, as well as a tool to
evaluate response to treatment. Furthermore, in clinical
trials for evaluation of antifungal drug efficacy, surro-
gate markers can be used to upgrade patients to a
diagnostic classification level with a higher degree of
certainty of infection. Commercial assays are available
for the detection of two circulating antigens: galacto-
mannan (GM) (Platelia Aspergillus, BioRad Labora-
tories) and (10/3)-b-D-glucan (BG) (Glucatell,
Associates of Cape Cod; Fungitec-G glucan detection
test, Seikagaku; Wako test, Wako Pure Chemical
Industries Ltd., Tokyo). These assays are now routinely
used in many centers throughout the world and are
commonly part of a diagnostic strategy in which they
are combined with other tests and procedures such as a
high resolution CT scan. The detection of fungal DNA
through PCR technology in blood or bronchoalveolar
lavage (BAL) fluid specimens also appears to be a
promising tool, but a standardized, commercialized
format is not available at present [1].
The different assays vary in the spectrum of fungi
they can detect as well as their performance character-
istics. Clinical validation studies have also shown
considerable variability in performance. For example,
the sensitivity and specificity of Aspergillus PCR-based
tests were found to range between 63%�/100% and
65%�/100%, respectively. By comparison, in GM tests
the sensitivity and specificity varied between 38%�/
100% and 81%�/100%, respectively [2]. The sensitivity
of GM detection appears to be lower in the more recent
studies compared to the results reported in earlier
investigations. While clinical studies of BG detection
have been limited, the data from a recent study
indicated the sensitivity and specificity to be 100%
and 90%, respectively [3]. The majority of these clinical
studies were performed with patients receiving treat-
ment for haematological malignancies, or undergoing
bone marrow or hematopoietic stem cell transplanta-
tion.
Detection of surrogate markers relies on intensive
blood sampling of high risk patients. Commonly,
monitoring is performed twice weekly during periods
of high risk or for patients with persistent fever not
responding to treatment with antibacterial agents.
Intensive prospective monitoring for GM and BG
antigens is required because circulating antigens have
Correspondence: P. E. Verweij, Department of Medical Microbiology,
University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB
Nijmegen, The Netherlands. Tel.: �/31 24 3614356; Fax: �/31 24
3540216; E-mail: [email protected]
– 2005 ISHAM DOI: 10.1080/13693780400025245
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been found to be present at a median of 8�/10 days
prior to diagnosis by conventional means [3,4].Furthermore, in patients with hematological malig-
nancy, antigen levels typically rise from negative to
positive over two to three days, so less frequent testing
would result in unnecessary delays in diagnosis. The
antigen remains positive in consecutive samples,
although the levels depend on the fungal burden in
the tissues.
Intensive sampling is required for detection of fungalDNA with PCR as it is usually detected in only a
minority of samples collected from patients with
invasive aspergillosis. In a recent study that prospec-
tively evaluated the use of nested Aspergillus species-
specific PCR, only 11%�/12% of systematically col-
lected blood samples obtained from 13 patients with
proven and probable invasive aspergillosis were PCR
positive [5]. This indicates that either the release andcirculation of fungal DNA is variable, or that the
concentration of fungal DNA in the blood is at the
lower limit of detection of the PCR assay used in this
investigation. Although the latter could be solved by
analysing a larger sample volume, there is a funda-
mental lack of understanding of the kinetics of release
and circulation of surrogate markers. Variables such as
the impact of conditions at the site of infection on therelease of markers or the effect of exposure to
antifungals are essential elements which need to be
investigated.
Variables that affect performance
A recent review listed the factors that interact with the
performance of Aspergillus antigen detection [2]. Theseincluded biological factors such as the site of infection,
exposure to antifungal agents, the presence of anti-
Aspergillus antibodies, as well as epidemiological
factors such as the specific patient population and the
interpretive cut-off points. Understanding the impact
of these variables is essential in explaining both false
positive and negative serological results.
A new cause for false positive reactivity is the use ofpiperacillin-tazobactam [6�/8]. Some manufactured lots
of this antibacterial agent have shown high reactivity
with the Platelia Aspergillus assay, creating false
positive results in patients treated with this drug [6�/
9]. Another possible etiology for false positive reactions
is a bacterial cell wall component associated lipo
teichoic acid (LTA), which has been suggested to cross
react with the EB-A2 monoclonal antibody used in thePlatelia Aspergillus ELISA due to similarity in mole-
cular structure with the galactomannan side chain
residues [10]. The LTA antigen is found in the cell
wall of Bifidobacteria , a group of bacteria known to be
present in high concentrations in the microbial flora offeces of neonates. Translocation of the antigen or
antigen bearing bacteria could account for the high
number of false positive ELISA reactivity in neonates,
which in one study was reported to be as high as 83.3%.
Although this appears to be a plausible explanation, it
remains to be proven and does not fully explain
reported false-positive reactions in older children.
Exposure to mould-active antifungal agents appearsto greatly influence the sensitivity of the Platelia
Aspergillus in bone marrow transplant recipients. The
sensitivity of the ELISA was only 16.7%�/20% in
patients receiving mould-active antifungal drugs as
compared to 80%�/87.5% in those not receiving these
compounds [11]. However, the specificity of the assay
was not affected by the use of antifungal drugs.
Although this subgroup analysis was based on a limitednumber of patients, it indicates that exposure to mould-
active antifungal compounds is an important variable
for the performance of the Platelia Aspergillus. This
may explain, in part, the wide range of sensitivity of the
assay (38%�/100%) reported in the literature. In order
to generate reliable data on performance characteristics
in clinical validation studies, the exposure of patients to
(prophylactically) administered antifungal drugs is anessential parameter that should be described in detail in
reports. In the same study, lowering the ELISA index
cut-off from 1.0 to 0.5 increased the median interval
from 1 to 10 days between test positivity and diagnosis
of invasive aspergillosis [11]. Other studies have con-
firmed the benefits of lowering the cut-off values [12],
as well as proposing variable cut-off levels [13].
Validating surrogate markers
The use of sensitive surrogate markers possesses
another problem related to the evaluation of perfor-
mance of the assays and clinical validation. In the
previously mentioned study by Buchheidt et al . [5], a
relatively high number of patients without invasive
aspergillosis tested positive. In 7% of these patients whowere classified as having ‘possible’ or ‘no’ invasive
aspergillosis, fungal DNA was detected in the blood.
Although the high number of ‘false’-positive samples
could be due to contamination, the likelihood of carry-
over contamination has decreased significantly over the
past years due to technical improvements such as the
use of automated PCR detection systems. An alter-
native explanation is the detection of true DNAemia inpatients with sub-clinical Aspergillus infection.
The European Organization for Research and Treat-
ment of Cancer/Mycosis Study Group (EORTC/MSG)
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classification system is based on patients with Asper-
gillus disease, e.g. clinical, microbiological and/orradiological evidence of disease [14]. However, before
Aspergillus disease becomes clinically evident, the
infection develops sub-clinically. Although patients
with sub-clinical infection will be classified as ‘no’ or
‘possible’ invasive aspergillosis, sensitive assays such as
PCR may detect circulating fungal DNA as an early
indicator of the presence of an infection. When the
PCR positive samples in the study of Buchheidt et al.
were subjected to quantitative PCR analysis there was
an increase of fungal burden with increasing certainty
of the diagnosis, i.e. the lowest burden was found in
patients classified as ‘no’ infection and the highest in
those with probable disease [5]. This finding supports
the concept of increasing fungal burden paralleling the
progression of infection from sub-clinical to clinical
disease [15]. Although our goal is to diagnose invasiveaspergillosis in patients as early as possible, detection of
fungal DNAemia in patients with sub-clinical infection
may complicate clinical validation studies.
Comparison of surrogate markers
Comparison of performance characteristics of surro-
gate markers remains difficult due to the significantvariability found among clinical validation studies
performed in different institutes or within the same
institute. A recent study attempted to compare real-
time PCR with GM detection with Platelia Aspergillus
and BG with the Wako test [12]. The test population
consisted of patients with hematological malignancy at
various stages of therapy including neutropenic and
non-neutropenic episodes. Unfortunately the samplingfrequency in this study was only once weekly and
patients received prophylactic treatment with either
mould-inactive fluconazole or mould-active itracona-
zole or amphotericin B. The performance of the
different markers was compared using receiver operat-
ing characteristic (ROC) analysis, in which the detec-
tion of GM proved to be the most sensitive when one
single positive reactive sample was considered anindicator of infection. When the same analysis was
performed defining two positive samples as indicators
of infection, the sensitivity of the GM detection
increased while that of the PCR and BG assays
decreased. This indicates that the GM assay has a
higher reproducibility than the other two assays.
Chronological comparison of the assays was possible
in only a limited number of episodes, although loweringof the cut-off value of the Platelia Aspergillus resulted
in earlier detection at a mean of 10 days before
diagnosis was made by conventional methods [12].
This study and similar ones published previously are
important in identifying which assays are the mostsensitive and specific, but also which is the first to
become positive during Aspergillus infection. Further-
more, assays may be more suitable to rule out the
presence of infection rather than detecting it. This will
help us to define the optimal testing strategy which may
differ for the various risk groups. It is unlikely that
performing all tests on all samples will become routine
practice due to the high costs and the absence ofclinical justification.
Cost-effectiveness studies should be performed in
order to prevent unnecessary diagnostic testing. Prob-
ability modifying plots have been used to determine
economically efficient sequences of testing without
losing clinical effectiveness [16]. This will tell us which
assay should be used first and what can be gained by
performing a second or even third assay. Comparedwith the costs of treatment, the costs of diagnostics are
relatively low [17]. This is one of many reasons to make
an effort to diagnose the presence of infection in every
suspected high risk patient, rather than treating them
empirically.
Conclusion
Advances in diagnostic testing for invasive aspergillosis
have been made. It is essential to understand the
kinetics of the various surrogate markers and to
identify factors that have significant impact on their
performance characteristics. Studies in these areas are
underway or have been recently published and will help
us to further improve our diagnostic strategies.
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