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Received: 12 August 2019    Revised: 20 September 2019    Accepted: 20 September 2019

DOI: 10.1111/myc.13012

ORIGINAL ARTICLE

Clinical application of Aspergillus lateral flow device in

bronchoalveolar lavage fluid of patients with classic risk factors
for invasive pulmonary aspergillosis

Kathleen A. Linder1,2 | Carol A. Kauffman1,2 | Shiwei Zhou1 | Marisa H. Miceli1

1
Division of Infectious Diseases, University
of Michigan, Ann Arbor, MI, USA
2
Infectious Diseases Section, Veterans
Affairs Ann Arbor Healthcare System, Ann
Arbor, MI, USA
Correspondence
Marisa H. Miceli, Division of Infectious
Diseases, University of Michigan Health
System, F4005 University Hospital South,
1500 E. Medical Center Dr. Ann Arbor, Ann
Arbor, MI 48109, USA.
Email: mmiceli@med.umich.edu
Funding information
The Veterans Education and Research
Association of Michigan
Abstract
Background: The diagnosis of invasive pulmonary aspergillosis (IPA) remains challeng‐
ing. We evaluated the performance characteristics of a newly formatted Aspergillus
lateral flow test, AspLFD, in bronchoalveolar lavage (BAL) fluid from patients with
classic risk factors for IPA.
Methods: Prospectively banked BAL samples from 14 patients with proven or prob‐
able IPA defined by EORTC/MSG criteria and 28 BAL samples from age‐matched
high‐risk patients without IPA were tested with AspLFD according to manufacturer's
directions. Results were read by two independent observers, and test performance
was calculated.
Results: Age, gender and underlying risk factors, except for neutropenia and haema‐
tological malignancy, were similar between IPA cases and controls. Seven patients
(50%) in the IPA group received a mould‐active agent within 5 days prior to bron‐
choscopy compared with only three patients (11%) in the control group, P = .004. Of
14 patients with proven/probable IPA, AspLFD was positive in 3 and negative in 9;
two tests yielded invalid results. All 28 control patients had a negative AspLFD test.
AspLFD showed low sensitivity (25%, 95% CI: 5.5% to 57.2%), but high specificity
(100%. (95% CI: 87.7% to 100%).
Conclusions: A positive AspLFD test in BAL fluid of patients with classic risk factors
for IPA could be useful to support the diagnosis of proven/probable IPA because of
its high specificity. However, as a stand‐alone test for IPA, the use of AspLFD is lim‐
ited by low sensitivity.

KEYWORDS
bronchoalveolar lavage, diagnostics, invasive aspergillosis, lateral flow device

1 |  I NTRO D U C TI O N
Diagnosis of invasive pulmonary aspergillosis (IPA) is challenging,
and delays in diagnosis contribute to increased mortality. Non‐in‐
vasive fungal biomarkers, such as the serum Aspergillus galactoman‐
nan (GM) assay, have proved useful in the diagnosis of IPA.1,2 When
performed on bronchoalveolar lavage (BAL) fluid, the GM assay has
shown increased sensitivity and specificity for IPA in patients who
have haematological malignancies and in those who have received a
hematopoietic cell transplant (HCT).1-3
The Aspergillus lateral flow device (AspLFD) is a newer non‐cul‐
ture‐based diagnostic test that utilises mouse monoclonal antibody
JF5 to detect an extracellular glycoprotein secreted during active
growth of Aspergillus species.4 This assay has been developed as

Mycoses. 2019;00:1–5. © 2019 Blackwell Verlag GmbH |  1

wileyonlinelibrary.com/journal/myc  
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2       LINDER et al.

a point of care test that can be performed on serum or BAL fluid. matching; but in some cases, matching was accomplished by using
Previous studies from our laboratory using a prototype lateral flow other classic risk factors.
device (LFD) demonstrated high specificity (94%) but low sensitivity The newly formatted AspLFD was used in accordance with the
(38%) on BAL samples from a population at high risk for IPA.5 AspLFD, manufacturer's protocol (OLM Diagnostics). BAL samples were
the newly formatted assay that has been approved for use in Europe, thawed, vortexed and centrifuged for 1 minute at 14 000 rpm, fol‐
6
was developed to enhance the performance of the LFD test. A lowing which 70 μL of the supernatant was applied to the device.
multicentre study of haematology patients using the AspLFD found After fifteen minutes, results were read independently by two in‐
a sensitivity of 71% and a specificity of 85% when comparing cases vestigators blinded to the patient's disease status and were scored
of proven or probable IPA with control patients without IPA.7 We as negative or positive; positive results were graded as +, ++ and
sought to determine the test performance of the AspLFD in BAL fluid +++ (Figure 1). If results were discordant, a third blinded investigator
from a broad population of patients with classic risk factors for IPA. was consulted as a tiebreaker. Results were considered invalid if the
control line was not positive after two separate runs. Of note, no
specimen required pretreatment per package insert instructions.
2 |  M ATE R I A L S A N D M E TH O DS We calculated the sensitivity and specificity of the AspLFD for
the diagnosis of proven/probable IPA. Differences in categorical
This study was performed at the University of Michigan Health variables or continuous numbers were analysed by Fischer's exact
System, and approval for the study was obtained from the University test or unpaired t test. All statistical analyses were completed using
of Michigan Institutional Review Board. Leftover BAL specimens SPSS software, version 25.0 (SPSS, Inc).
from all bronchoscopies performed on adult patients between
11/2015 and 2/2018 were collected prospectively. BAL fluid was
stored at −70°C in an established BAL repository in the Infectious 3 | R E S U LT S
Diseases Laboratory at the VA Ann Arbor Healthcare System.
The medical records of all patients whose BAL fluid was collected A total of 795 patients had samples stored in the BAL repository and
were reviewed to identify adult patients ≥ 18 years of age with classic clinical data recorded in the REDCap system. Sixteen of these pa‐
risk factors for IPA. Classic risk factors were defined as haematolog‐ tients had IPA: two proven, 12 probable and two possible. The latter
ical malignancy, neutropenia (absolute neutrophil count ≤ 500 cells/ two patients were excluded from further analysis.
μL for ≥ 10 days at time of BAL), solid organ or HCT recipient, inher‐
ited severe immunodeficiency, HIV with CD4 count ≤ 200 cells/μL,
use of T‐cell immunosuppressive agents within 90 days of BAL or use TA B L E 1   Demographics of patients with invasive pulmonary
aspergillosis (IPA) and control patients who had bronchoalveolar
of corticosteroids at a dose equivalent to ≥ 0.3 mg/kg of prednisone
lavage samples tested with AspLFD
daily for ≥ 3 weeks at time of BAL. Receipt of a mould‐active antifun‐
gal agent within 5 days prior to bronchoscopy was noted. Previously IPA cases Controls
(n = 14) (n = 28)
obtained results from serum and BAL Aspergillus GM (PlateliaTM
Aspergillus EIA, Viracor‐IBT Laboratories) and respiratory fungal cul‐   Number (%) Number (%) P‐value
tures were recorded. GM results with optical density index (ODI) Mean age (years + SD) 52.7 ± 17.5 53.6 ± 15.6 .86
values ≥ 0.5 were considered positive. All data were entered into a
Female 6 (43) 8 (29) .35
REDCap research database.
Classic risk factors for IPA
The diagnosis of IPA was determined using EORTC/MSG cri‐
T‐cell depleting agent 9 (64) 20 (71) .64
teria; cases were categorised as proven, probable, possible or no
Solid organ transplant 4 (29) 14 (50) .19
IPA.8 Patients with proven or probable IPA were matched 1:2 by
High dose corticosteroidsa 4 (29) 11 (39) .49
age within 5 years and by risk factors with controls who did not
have IPA. Whenever possible, the same risk factors were used for Haematological malignancy 6 (43) 3 (11) .02
Neutropeniab 3 (21) 0 (0) .01
Allogeneic HCT 0 (0) 2 (7) .31
Additional features
Anti‐mould exposurec 7 (50) 3 (11) .004

Abbreviations: COPD, chronic obstructive pulmonary disease; HCT,

hematopoietic cell transplant.
a
Defined as a dose equivalent to ≥ 0.3 mg/kg prednisone daily
for ≥ 3 weeks at the time of the BAL.
b
Defined as an absolute neutrophil count ≤ 500 cells/μL for ≥ 10 days at
the time of the BAL.
F I G U R E 1   Examples of positive results graded as +, ++ and +++ c
Defined as receipt of a mould‐active antifungal agent within 5 days
and a negative result with the AspLFD assay prior to bronchoscopy.
 LINDER et al.

TA B L E 2   Patients with proven/probable invasive pulmonary aspergillosis (IPA)

Age/Sex Host Factors Mould‐active agenta IPA BAL/sputum culture BAL GMb Serum GMb BAL AspLFD

60M T‐cell immunosuppression Voriconazole Probable A fumigatus 1.36 Not done Positive (+++)
69F Neutropenia, corticosteroids, T‐cell None Probable Negative 2.80 Negative Positive (+)
immunosuppression
79F Haematological malignancy Voriconazole Probable Aspergillus spp. Not done Negative Positive (+)
c
42M Corticosteroids (Cushing's syndrome) Voriconazole, Liposomal ampho‐ Proven Negative 0.08 Negative Negative
tericin B
32F Corticosteroids, T‐cell None Provenc A fumigatus 0.68 Negative Negative
immunosuppression
60M Haematological malignancy, T‐cell None Probable A fumigatus 0.21 Not done Negative
immunosuppression
22M Neutropenia, Haematological malignancy Voriconazole, Micafungin Probable Negative 6.21 Negative Negative
42M Lung Tx, T‐cell immunosuppression None Probable A fumigatus 0.15 Not done Negative
62M Lung Tx, T‐cell immunosuppression None Probable A fumigatus 0.12 Not done Negative
23F Lung Tx, T‐cell immunosuppression None Probable A fumigatus 0.21 Negative Negative
66M Haematological malignancy, T‐cell Isavuconazole Probable Negative 0.11 1.75 Negative
immunosuppression
63F Lung Tx, T‐cell immunosuppression Voriconazole Probable A fumigatus (sputum) 0.14 Negative Negative
58F Corticosteroids None Probable Negative 7.6 Negative Invalidd
60M Neutropenia, Haematological malig‐ Voriconazole, Liposomal ampho‐ Probable Negative 2.3 Negative Invalidd
nancy, T‐cell immunosuppression tericin B

Abbreviation: AspLFD, Aspergillus lateral flow device; GM, galactomannan; Tx, transplant.
a
Patients received a mould‐active antifungal agent within 5 days prior to bronchoscopy.
b
Expressed as optical density index (ODI); values ≥ 0.5 ODI defined as positive.
c
Autopsy‐proven aspergillosis.
d
Results were considered invalid if the control line was not positive on two separate tests.
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4       LINDER et al.
The mean age of the 14 patients with proven/probable IPA was
52.7 ± 17.5 years, and 6 (43%) were female. Of the 28 control pa‐
tients without IPA, the mean age was 53.6 ± 15.6 years, and 8 (29%)
were female. Comorbid conditions and risk factors for IPA were sim‐
ilar for both groups except for haematological malignancy and neu‐
tropenia, which were more common in patients with IPA (Table 1).
Seven patients (50%) in the IPA group were on a mould‐active anti‐
fungal agent within the 5 days prior to bronchoscopy compared with
only 3 patients (11%) in the control group, P = .004.
Among the 14 patients with proven/probable IPA, BAL fluid in 7
patients and sputum in 1 patient yielded Aspergillus species; six pa‐
tients had a BAL GM ≥ 0.5 ODI, 5 of which were ≥ 1.0 ODI (Table 2).
computarised tomography scans were performed in 13 patients;
nodules were present in 10 patients, tree‐in‐bud changes in 3 and a
halo sign, a cavitary lesion and a mycetoma in one patient each. One
patient, who expired and had autopsy‐proven IPA, did not have a
scan performed before death.
For the 14 patients with proven/probable IPA, AspLFD was pos‐
itive in 3 and negative in 9 (Table 2). Four of the 7 patients on a
mould‐active agent had negative AspLFD test results, and 2 had a
positive AspLFD test result. Two patients with probable IPA had in‐
valid AspLFD results because the control line did not appear, and
these results were excluded from further analysis. All 28 control pa‐
tients had a negative AspLFD result. Inter‐grader concordance was
high; an adjudicator was needed for only three samples.
Sensitivity of the AspLFD in BAL fluid was 25% (95% CI: 5.5% to
57.2%), and specificity was 100% (95% CI: 87.7% to 100%).
4 |  D I S CU S S I O N
The AspLFD is an attractive option for testing for IPA, as it can be
done quickly as a point of care test and can be used with either
serum or BAL fluid. However, test performance has varied among
different laboratories. Initial studies showed promising results using
a prototype LFD.5,9-13 These studies each involved different patient
populations, but in general, the prototype LFD assay was noted to
have specificity > 90%. However, sensitivity varied from 38% to
94%. Reasons for this wide discrepancy could, in part, be related to
different populations that were studied and to variations in study
design. For instance, in several studies, a modified definition of IPA
was used to enable comparison of the LFD with other biomarkers,
such as GM.5,11
The newly formatted AspLFD was developed to improve the per‐
formance of the prototype LFD assay. Five prior reports using the
AspLFD noted sensitivity that varied from 46% to 78% and speci‐
ficity that varied from 66% to 100%.6,7,14-16 When using the AspLFD
assay in a population with classic risk factors for IPA, we found im‐
provement in sensitivity and specificity over the prototype LFD, as
6
previously had been noted by Hoenigl et al.
Several prior studies have noted that exposure to mould‐ac‐
tive antifungal agents decreased the sensitivity of the prototype
LFD assay. 5,17,18 Additionally, the prototype LFD assay performed
better in studies evaluating its use in patients who had received a
solid organ transplant than in patients with haematological malig‐
nancies, which has been postulated to be due to the use of prophy‐
laxis with mould‐active antifungal agents in the latter group.18 One
study has addressed this issue using the newly formatted AspLFD
assay.7 In the subgroup of patients who had received empirical
mould‐active agents prior to AspLFD testing, the sensitivity of the
AspLFD assay decreased from 71% to 47%. In our study, 50% of pa‐
tients with proven/probable IPA had received a mould‐active anti‐
fungal agent within the 5 days prior to the collection of BAL fluid;
this may have led to decreased sensitivity of the AspLFD assay.
A similar phenomenon has been described previously for the GM
assay and for PCR.19-21
Similar to other qualitative tests, interpretation of AspLFD results
is operator dependent. In our study, overall concordance among in‐
vestigators was high, but Inter‐grader variability could contribute to
differences in performance that have been noted among different
studies. A recent large multicentre study used a digital reader in
addition to visual assessment of test results and noted use of the
reader enhanced the ability to detect lines.7 The use of such a digital
reader should be explored further.
Some authors have suggested that a dual testing approach com‐
bining an LFD assay and a GM assay could improve sensitivity for the
diagnosis of IPA.5,22,23 Studies using this combined testing approach
with the prototype LFD and GM are contradictory, with some show‐
ing increased sensitivity and others not. One group used this ap‐
proach with the newly formatted AspLFD and reported enhanced
sensitivity.15
Limitations of this study include the single‐centre retrospective
nature and the small number of patients with proven or probable
IPA. Our samples had been frozen at −70°C before use, and al‐
though, unlikely, this possibly could have decreased the sensitivity
of the AspLFD test.
Benefits of the AspLFD include the ease of use and high inter‐
observer agreement regarding endpoints, which are important for
point of care tests. A positive test is highly specific for IPA, but sen‐
sitivity is low, which would not allow its use as a stand‐alone test for
IPA. Further studies are warranted to determine the ultimate role of
this assay in the clinical setting.
AC K N OW L E D G E M E N T S
This study was supported by the Veterans Education and Research
Association of Michigan.
C O N FL I C T S O F I N T E R E S T
Carol A. Kauffman is a member of the Data Safety Monitoring Board
for CIDARA. Marisa H. Miceli is a consultant for Astellas and a mem‐
ber of the Data Review Committee for Scynexis. Shiwei Zhou and
Kathleen Linder have no conflicts to declare.
LINDER et al.
AU T H O R C O N T R I B U T I O N
KAL : collected data, tested specimens and wrote manuscript. CAK:
designed study, tested samples, and wrote manuscript. SZ colelcted
data, MHM: concept and design design, sample testing, data analysis
and manuscript writing.
ORCID
Marisa H. Miceli  https://orcid.org/0000-0002-3175-0512
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How to cite this article: Linder KA, Kauffman CA, Zhou S,
Miceli MH. Clinical application of Aspergillus lateral flow
device in bronchoalveolar lavage fluid of patients with classic
risk factors for invasive pulmonary aspergillosis. Mycoses.
2019;00:1–5. https​://doi.org/10.1111/myc.13012​