JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2004, p. 4154–4157 Vol. 42, No.

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0095-1137/04/$08.00⫹0 DOI: 10.1128/JCM.42.9.4154–4157.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Diagnosing Invasive Aspergillosis during Antifungal Therapy by PCR

Analysis of Blood Samples
Cornelia Lass-Flörl,1* Eberhard Gunsilius,2 Günther Gastl,2 Hugo Bonatti,3 Martin C. Freund,4
Andreas Gschwendtner,5 Gabriele Kropshofer,6 Manfred P. Dierich,1 and Andreas Petzer3
Department of Hygiene and Social Medicine1 and Department of Pathological Anatomy,5 University of Innsbruck, and
Department of Haematology and Oncology,2 Department of Surgery,3 Department of Radiology I,4 and
Department of Pediatric Oncology,6 University Hospital Innsbruck, Innsbruck, Austria
Received 4 March 2004/Returned for modification 11 April 2004/Accepted 6 June 2004

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We evaluated the value of Aspergillus PCR as a tool for diagnosing invasive aspergillosis from whole-blood
samples during antifungal therapy. In a 3-year study, 36 patients receiving antifungal therapy due to chest
radiographic findings highly suggestive of fungal pneumonia were evaluated. The PCR results from whole-
blood samples were compared to those obtained from bronchoalveolar lavage fluids and/or tissue specimens.
A total of 205 whole-blood samples, 15 fine-needle aspirations or tissue biopsy specimens, and 21 bronchoal-
veolar lavage fluids and tracheal secretions were analyzed using PCR. Of the 36 patients, 15 had proven, 9 had
probable, and 12 had possible invasive Aspergillus infection according to European Organization for Research
and Treatment of Cancer/Mycosis Study Group definitions. For patients with proven infection the sensitivity
values of PCR in lung and blood samples were 100 and 40%, respectively. The negative predictive value of blood
monitoring under conditions of antifungal treatment was 44%. Clearance of fungal DNA from blood was
associated with resolution of clinical symptoms in six of nine patients with proven infection. Repeated positive
PCR results for Aspergillus were associated with fatal outcome, as three of six patients died. For patients with
probable infection the sensitivity values of PCR in lung fluid and blood were 66 and 44%, respectively. The
benefit of PCR diagnosis using whole-blood samples is limited when sampling takes place after treatment has
been started. Performance of Aspergillus PCR using tissue samples is recommended in addition to microscopic
examination and culture technique for sensitive detection of fungal infection.

Invasive aspergillosis (IA) is increasingly recognized in im- in patients at risk. Hence, positive results became negative
munocompromised hosts (9). Patients with prolonged and shortly after commencement of antifungal therapy and did not
deep granulocytopenia following chemotherapy for hemato- correlate with clinical responsiveness to treatment.
oncologic disorders or allogeneic bone marrow transplant re- This study examined the diagnostic value of PCR of whole-
cipients are particularly at risk (2, 21). The case fatality rate of blood samples during antifungal therapy. Evaluation was per-
IA approaches 100% and results at least partly from difficulties formed prospectively over a 3-year period for patients with
in obtaining a reliable diagnosis at an early stage of the disease, proven, probable, or possible aspergillosis. The PCR results of
often leading to a fatal delay in adequate therapy (1). No whole-blood samples were compared to those obtained from
method has proven sufficiently sensitive and specific to allow bronchoalveolar lavage (BAL) fluid and/or tissue specimens.
diagnosis at an early stage. Culture detection is often delayed,
and blood culture results are rarely positive for patients with
MATERIALS AND METHODS
IA (10). The Aspergillus galactomannan (GM) enzyme-linked
immunosorbent assay is presently the most promising test for Patients. A prospective study conducted between January 2000 and September
2003 evaluated 36 patients with hematological malignancies or solid-organ trans-
diagnosis of IA (20). However, Herbrecht et al. (14) observed
plantations (median age, 44 years [range, 19 to 78 years]) who were undergoing
that the GM assay seems less sensitive than previous studies antifungal therapy due to chest radiographic findings highly suggestive of As-
have suggested. Buchheidt et al. (8) compared PCR to the GM pergillus pneumonia. Blood samples, fine-needle aspirations, tissue biopsies,
assay in a prospective study and found that PCR was superior BAL, and tracheal secretion (TS) fluid specimens were investigated by culture
to the GM assay with respect to sensitivity (63.6 versus 33.3%). technique, histopathologic examination, and PCR to confirm invasive fungal
disease. Blood samples were taken twice a week, and at least three samples were
So far, the detection of circulating fungal DNA has been ad- required for inclusion in the study. Patients received intensive myelosuppressive
vocated as a promising, rapid, and more sensitive diagnostic or immunosuppressive chemotherapy for hematological malignancies (n ⫽ 25) or
tool for overcoming these drawbacks (5–7, 11, 12, 25). long-term therapy with corticosteroids (n ⫽ 2) or had had an organ transplan-
In a previous study Lass-Flörl et al. evaluated the value of tation (n ⫽ 9).
All patients received amphotericin B (Bristol Meyer Squibb, Vienna, Austria)
twice-weekly screening for circulating fungal DNA by means of
(1.0 to 1.5 mg/kg of body weight/day) or intravenous voriconazole (Vfendt;
PCR of whole-blood samples (15). Our results indicate the Pfizer, Austria) (4 mg/kg/12 h or 200 mg/day orally), liposomal amphotericin B
potential usefulness of PCR when screening for Aspergillus spp. (Ambisome; Gilead, Vienna, Austria) (3 to 5 mg/kg/day), caspofungin (Cancidas;
MSD, Vienna, Austria) (70 mg/day), itraconazole (Sporanox; Jannson Cilag,
Vienna, Austria) (200 mg/12 h), or several combinations of the above-mentioned
* Corresponding author. Mailing address: Department of Hygiene antifungal drugs at the time of blood monitoring. For all patients dying from
and Social Medicine, Medical University Innsbruck, Fritz Pregl Str. 3, pulmonary infections, autopsy samples were collected for microscopy, fungal
6020 Innsbruck, Austria. Phone: 43 512 507 3425. Fax: 43 512 507 2870. culture, and histopathologic evaluation.
E-mail: Cornelia.Lass-Floerl@uibk.ac.at. In addition, before starting antifungal treatment blood samples were drawn

4154
VOL. 42, 2004 Aspergillus DETECTION IN BLOOD 4155

TABLE 1. PCR results for patients with proven aspergillosis and who were undergoing antifungal therapy
Result ofe:
Outcome
Patient a
Age/sex Underlying disease or cause due to culture of lung Microscopic PCR PCR before start of
no. HRCTd
infection specimens examination antifungal therapyc
Bloodb Specimenc

1 27/M ALL Dead A. fumigatus Pos Neg Pos Pos Not done
2 47/M AML Alive A. fumigatus Pos Neg Pos Pos Not done
3 31/M ALL Dead A. terreus Pos Pos (2) Pos Pos Pos (2)
4 51/M AML Alive Neg Pos Neg Pos Pos Not done
5 59/F Organ transplantation Dead A. fumigatus Pos Pos (1) Pos Pos Pos (1)
6 61/M Organ transplantation Alive Neg Pos Neg Pos Pos Pos (1)
7 51/M Organ transplantation Alive A. terreus Pos Neg Pos Pos Not done
8 63/F Organ transplantation Alive Neg Pos Pos (2) Pos Pos Not done
9 49/M NHL Dead Neg Pos Neg Pos Pos Pos (2)
10 70/F MDS Alive Neg Pos Pos (1) Pos Pos Neg

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11 29/F ALL Dead Neg Pos Neg Pos Pos Pos (1)
12 67/M Aplastic anemia Dead A. terreus Pos Pos (1) Pos Pos Not done
13 53/M Solid tumor Alive A. fumigatus Pos Pos (1) Pos Pos Pos (1)
14 27/M ALL Alive A. terreus Pos Neg Pos Pos Neg
15 69/F MDS Alive Neg Pos Neg Pos Pos Not done
a
ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; NHL, non-Hodgkin’s lymphoma; MDS, myelodysplastic syndrome.
b
Values in parentheses are numbers of positive PCR samples.
c
Specimens originated from lung biopsy or fine-needle aspiration.
d
Positive high-resolution computer tomography results indicate findings consistent with invasive aspergillosis.
e
Pos, positive; Neg, negative.

from 15 patients (with proven, probable, or possible aspergillosis) in case fever proven, 9 showed probable, and 12 showed possible invasive
developed during broad-spectrum antibacterial therapy. Four BAL samples, Aspergillus infection, as shown in Table 1, Table 2, and Table 3.
three lung specimens, and seven blood samples from immunocompetent patients
with proven bacterial infection served as a negative control.
In patients with proven infection the sensitivity of PCR in
Definitions. IA was assessed on the basis of EORTC/MSG criteria (3). Proof lung and blood samples was 100 and 40%, respectively. Spec-
of infection was based on histopathologic or cytopathologic examinations show- ificity was 100%. The negative predictive value of blood mon-
ing hyphae from needle aspirations or biopsy specimens with evidence of asso- itoring with antifungal treatment was 44%. In patients with
ciated tissue damage or a positive culture for a sample obtained by sterile
probable infection the sensitivity of PCR in lung fluid and
procedure from a normally sterile site and a clinically or radiologically abnormal
site consistent with infection. Patients with a probable invasive infection were blood was 66 and 44%, respectively. Specificity was 100%. The
characterized by at least one host factor criterion, one microbiological criterion, negative predictive value of blood monitoring with antifungal
and one major clinical criterion (or two minor clinical criteria). therapy was 58%. Positive PCR results correlated to 100%
Patients with a possible invasive infection were characterized by at least one with the microscopic presence of fungal hyphae in patients
host factor criterion and one microbiological criterion or one major clinical
criterion (or two minor clinical criteria).
with proven infection. Three patients with culture-positive
PCR assay. DNA extraction of 10 ml of EDTA-anticoagulated blood was samples showed negative BAL-TS PCR results. By contrast,
performed using recombinant lyticase (Sigma, Vienna, Austria) and a QIAmp seven patients showed positive lung specimen PCR results, and
tissue kit (QIAGEN, Vienna, Austria) as previously described (11). In addition, yet culture results were negative.
biopsy specimens were treated with lyticase and proteinase K and beaten with
All patients received antifungal therapy for at least 3.1 days
glass beads. A highly conserved sequence of the multicopy 18S rRNA of various
fungal pathogens was amplified by PCR using specific primers. Primers (Roth, (mean; range, 2 to 15 days) before diagnosis of blood by PCR
Graz, Austria) (5⬘-ATT GGA GGG CAA GTC TGG TG and 5⬘-CCG ATC was performed. Biopsy specimens were taken between 5 and 31
CCT AGT CGG CAT AG) bind to conserved regions of this 18S rRNA. A total days after the start of antifungal therapy.
of 34 cycles of repeated denaturation (94°C for 30 s), annealing (62°C for 1 min), Of the patients with proven aspergillosis and PCR positivity
and extension (72°C for 2 min) were applied. Amplicons were detected by
PCR–enzyme-linked immunosorbent assay using digoxin-labeled oligonucleotide
during treatment, 50% (three of six) died; of those, 33% (three
(Roth) (5⬘-CAT GGC CTT CAC TGG CTG TGG GGG GAA CCA) for of nine) exhibited a negative PCR signal. Similar results were
Aspergillus spp. and antidigoxigenin antibodies conjugated with alkaline phos- observed in patients with probable infection, as shown in Table
phatase (Boehringer Mannheim, Vienna, Austria) as described earlier (18). 2. Of patients with proven aspergillosis, six showed positive
Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus nidu-
PCR results before starting antifungal therapy; four of those
lans were detected with these genus-specific oligonucleotide probes. Negative
and positive probes were routinely used for quality control. Several PCR-nega- patients died from IA. No inhibition of PCR signal was ob-
tive blood samples from patients with proven aspergillosis were retested by served in the various samples tested.
adding 20 pg of A. fumigatus bacteria to the samples to detect PCR inhibition.

DISCUSSION
RESULTS
We evaluated the value of Aspergillus PCR as a tool for
A total of 205 whole-blood samples (mean, 4.7 samples/ diagnosing IA from whole-blood samples during antifungal
patient; range, 3 to 8), 15 fine-needle aspirations or tissue therapy. The sensitivity of sequential blood samples in patients
biopsy specimens, and 21 BAL or TS fluid specimens from 36 with proven infection was 40% and in patients with probable
patients were analyzed using PCR. Of the patients, 15 showed infection was 44%; the negative predictive values were 44%
4156 LASS-FLÖRL ET AL. J. CLIN. MICROBIOL.

TABLE 2. PCR results in patients with probable aspergillosis and undergoing antifungal therapy
Result ofd:
Outcome
Patient a
Age/sex Underlying disease or cause due to Culture of Microscopic PCR PCR before start of
no. HRCTc
infection BAL and/or TS examination antifungal therapyb
Bloodb BAL or TS

1 25/M AML Dead A. fumigatus Not done Neg Pos Pos Not done
2 39/M CLL Alive A. fumigatus Pos Pos (1) Pos Pos Not done
3 77/M MDS Dead A. terreus Pos Neg Pos Pos Pos (2)
4 31/F Solid tumor Alive A. fumigatus Not done Neg Neg Pos Not done
5 65/M Aplastic anemia Dead A. terreus Pos Pos (1) Pos Pos Pos (1)
6 47/F Organ transplantation Dead A. fumigatus Not done Pos (2) Neg Pos Not done
7 51/M Organ transplantation Alive A. terreus Not done Neg Neg Pos Pos (2)
8 38/F Organ transplantation Alive A. fumigatus Pos Pos (2) Pos Pos Not done
9 28/F ALL Dead A. terreus Pos Neg Pos Pos Not done

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a
AML, acute myeloid leukemia; CLL, chronic lymphoid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoid leukemia.
b
Values in parentheses are numbers of positive PCR samples.
c
Positive high-resolution computer tomography results indicate findings consistent with invasive aspergillosis.
d
Pos, positive; Neg, negative.

and 58%, respectively. Similar data were found by others when of disease and thus more consistent results (5). Loeffler et al.
using nested PCR (8, 13, 26). Because PCR can be inhibited by (19) reported that the sensitivity of plasma PCR was lower
the presence of antifungals, we spiked several PCR-negative than that of PCR performed with whole-blood samples. Ana-
blood samples and showed positive PCR results. Thus, it can lyzing blood samples is clinically applicable, because the sam-
be assumed that antimycotic treatment is in part responsible ples can be obtained repeatedly and by noninvasive means. So
for the clearance of fungi from blood to nondetectable levels far, repeated positive PCR results for Aspergillus have been
whereas clearance from tissue does not occur (15). This as- associated with fatal outcome, as three out of six patients died,
sumption is supported by PCR-negative blood sample results and a finding of several negative PCR blood results does not
combined with positive-testing tissue sample results for our exclude the presence of aspergillosis. Clearance of fungal DNA
patients. In addition, the half-life of circulating DNA is short, from blood was associated with resolution of clinical symptoms
probably less than 5 min, and a positive signal is observed only in six of nine patients with proven infection. However, to assess
when the fungal burden is large enough (23). Yet Loeffler et al. this PCR assay as a means of monitoring responding and non-
(17) postulate a correlation between high fungus load in tissue responding patients, further studies are needed. Screening for
and the presence of fungal DNA in blood. In their study, and diagnosis of aspergillosis prior to treatment gives better
Aspergillus DNA was detected in only 25% of the blood sam- sensitivity, as shown in the literature (13, 16). Four patients
ples from infected animals. Mice whose blood became PCR showing PCR positivity prior to antifungal treatment died;
positive showed a mean fungus load in the lung 10 times higher whether such individuals might benefit from immediate anti-
than that in mice whose blood remained PCR negative. Similar fungal therapy remains to be investigated in more detail. How-
differences in mean fungus loads could matter in humans. ever, one of the major drawbacks to this application of As-
A PCR assay that detects circulating DNA in serum can pergillus PCR is the high rate of transient Aspergillus fungemia
possibly give a better correlation with fungal load and severity without evidence of IA (4).

TABLE 3. PCR results in patients with possible aspergillosis and undergoing antifungal therapy
Result ofd:
Outcome
Patient a
Age/sex Underlying disease or cause due to Culture of Microscopic PCR PCR before start of
no. HRCTc
infection BAL and/or TS examination b antifungal therapyb
Blood BAL or TS

1 44/M HD Alive Neg Neg Neg Neg Pos Pos (2)

2 32/M Solid tumor Alive Neg Neg Neg Pos Pos Not done
3 43/F Multiple myeloma Alive Neg Not done Pos (2) Neg Pos Not done
4 41/M NHL Alive Neg Neg Neg Pos Pos Pos (1)
5 47/M HD Alive Neg Not done Pos (1) Neg Pos Not done
6 62/M Organ transplantation Alive Neg Not done Neg Neg Pos Not done
7 62/F Organ transplantation Alive Neg Neg Neg Neg Pos Pos (1)
8 43/F Solid Tumor Alive Neg Neg Pos (2) Pos Pos Not done
9 51/F Solid Tumor Alive Neg Not done Neg Pos Pos Pos (2)
10 59/M ALL Dead Neg Neg Neg Pos Pos Not done
11 27/F ALL Alive Neg Not done Neg Neg Pos Not done
12 52/M CML Alive Neg Neg Pos (1) Neg Pos Not done
a
HD, Hodgkin’s disease; NHL, non-Hodgkin’s lymphoma; ALL, acute lymphoid leukemia; CML, chronic myeloid leukemia.
b
Values in parentheses represent the numbers of positive PCR samples.
c
Positive high-resolution computer tomography results indicate findings consistent with invasive aspergillosis.
d
Pos, positive; Neg, negative.
VOL. 42, 2004 Aspergillus DETECTION IN BLOOD 4157

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