Received Date : 04-Jan-2015

Revised Date : 08-Mar-2015

Accepted Date : 04-Apr-2015

Article type : Review

Diagnosis of Invasive Aspergillosis: Recent Developments and

Ongoing Challenges

Marios Arvanitis1,2,3, MD, Eleftherios Mylonakis1,2, MD, PhD

Affiliations: 1Infectious Diseases Division, Rhode Island Hospital, Providence, RI;

2
Warren Alpert Medical School of Brown University, Providence, RI; 3Internal
Medicine Department, Boston Medical Center, Boston, MA

Corresponding Author: Eleftherios Mylonakis, Infectious Diseases Division, Rhode

Island Hospital, 593 Eddy Street, POB, 3rd Floor, Suite 328/330, Providence, RI,
02903, [emylonakis@lifespan.org], Tel: 401-444-7856 / Fax: 401-444-8179

Abstract

Background: Invasive aspergillosis is an infection with high morbidity and mortality

that affects mostly immunocompromised individuals. Early identification and targeted
treatment of the infection is essential to improve survival of affected patients. The
purpose of our review is to highlight the most recent developments in diagnosis and
screening for invasive aspergillosis along with the challenges associated with the
development and validation of novel diagnostic approaches.

Methods: Ovid MEDLINE and The Cochrane library were searched for studies that
evaluated serologic, molecular and novel methodologies for the diagnosis of invasive
aspergillosis

This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/eci.12448
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Results: Traditional diagnostic approaches, such as histopathology and culture, are
still considered the gold standard but lack sufficient sensitivity. Newer serologic
techniques, such as galactomannan and beta-glucan, have already been incorporated
in clinical guidelines but recent evidence suggest that their performance might be
limited in certain clinical settings. Molecular methods, such as the Aspergillus spp.
polymerase chain reaction (PCR), have not yet found their place in clinical practice
mainly due to lack of standardization. Novel methodologies, such as volatile organic
compound detection and lateral flow devices, have recently been developed and
promise non-invasive and rapid diagnosis of aspergillosis, while diagnostic algorithms
that incorporate both galactomannan and PCR have proven to be effective in early
randomized trials as screening methods and can reduce the use of antifungal agents.

Conclusions: Diagnosis of invasive aspergillosis remains challenging. Novel

methodologies and the standardization of Galactomannan and PCR might provide
more reliable diagnostic tools in the future.

Introduction

Fungi are abundant in our environment. Most of them are harmless to humans, while
others can cause infections that range from simple skin infections to severe systematic
illness that can lead to multiorgan failure and death. Prominent among these
pathogenic fungi are Aspergillus spp. Diseases caused by Aspergillus spp. belong to a
spectrum from allergic bronchopulmonary aspergillosis to invasive aspergillosis (IA).
IA is an opportunistic infection that affects severely immunocompromised individuals
and mostly people with hematologic malignancies or organ transplants. Early
diagnosis and identification of the infection is paramount to decrease the high
morbidity and mortality associated with the disease. The ideal diagnostic test has yet
to be discovered, but several new serologic and molecular approaches show promising
findings.

Challenges in the diagnosis of IA

The development of an accurate and reliable diagnostic test for IA remains the holy
grail of diagnostic mycology. Cultures of respiratory specimens obtained by sputum
induction or bronchoalveolar lavage (BAL) along with histopathologic detection of
the fungus on biopsy specimens constitute the gold standard methods for the diagnosis

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of the infection. However, these methods are far from ideal. Biopsy specimens are not
always obtained, nor feasible in critically ill individuals with bone marrow
suppression and multiple comorbidities (1). Further, cultures of respiratory specimens
have relatively low positive predictive value (about 72% in one study (2)) which can
decrease even more when testing non-hematology patients (3) or patients who are
already receiving antifungal agents. In addition, growth of Aspergillus spp. from
respiratory specimens does not de facto mean an infection as many patients can
become colonized with mold (3, 4).

In the absence of a reliable microbiologic identification, the diagnosis of IA often

depends on radiographic findings in patients who are at high risk for the disease.
Indeed, computed tomography (CT) findings like the halo sign or the air-crescent sign
have traditionally been known as characteristic of the disease in the appropriate
clinical setting. However, we now know that these findings can only be suggestive of
the infection and are not specific enough to be considered pathognomonic. Indeed,
other invasive fungal infections, like Scedosporium spp. or Cryptococcus spp., can
have similar CT findings as can bacterial infections like Pseudomonas spp. (5).
Recent evidence suggest that positron emission tomography combined with CT may
be able to lead to higher specificities for IA (6) and could potentially differentiate
between invasive infection and colonization or chronic non-invasive forms of the
disease (7, 8). However, these data come from generally small studies and have yet to
be validated in large cohorts to obtain reliable performance estimates.

As the need for an accurate and easy to perform diagnostic test for IA has yet to be
satisfied, it comes as no surprise that development and validation of newer diagnostic
modalities are currently in the spotlight of scientific research. Unfortunately, the lack
of a reliable gold standard constitutes a major obstacle for the validation of new
diagnostic methods. Indeed, in the absence of an accurate comparator, it is very
difficult to estimate the performance estimates of a new test. Most trials and
guidelines have tried to circumvent this problem by using the criteria developed by
the European Organization for the Research and Treatment of Cancer/ Mycoses Study
Group (EORTC/MSG) (9, 10). These criteria were developed specifically for this
purpose and divide the patients suspected to have IA into four categories (proven,
probable, possible and unlikely IA), based on risk factors, radiographic and
microbiological criteria.

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While the EORTC/MSG criteria are now widely used as the reference standard in
clinical trials, their use has some disadvantages. The main problem relies on the
classification of some patient cases as possible IA. This classification applies to
individuals who have risk factors and suggestive radiographic findings but do not
have one of the microbiological criteria that define a probable IA case. While most
clinical studies group possible IA cases together with unlikely IA cases as true
negatives, and while it has been suggested that the possible IA classification should
not be used to make treatment decisions (11), it is an unavoidable occurrence that
some of the patients classified as possible IA will in fact have the disease. This
misclassification of some IA cases will in turn lead to miscalculation of the
performance estimates of a diagnostic test in development. Given the absence of any
better reference standard, the use of the EORTC/MSG criteria in Aspergillus spp.
research is here to stay. However, their limitations should be realized and efforts to
refine and improve these criteria should be made based on the evolving literature
behind IA diagnosis.

Recent developments in the diagnosis of IA

IA identification is a rapid growing field and several novel serologic and molecular
diagnostic technologies have relatively recently been discovered and validated (Table
1). Some of them, like galactomannan (GM) and beta glucan, have already been
clinically evaluated and are now widely used in clinical practice, while others, like the
Aspergillus polymerase chain reaction (PCR) have yet to be implemented in clinical
laboratories.

Galactomannan

The galactomannan antigen is a component of the cell wall of Aspergillus spp. (12).
An assay that can detect the GM antigen in clinical specimens has been recently
developed and validated and now holds a very prominent position in IA diagnostics as
it has been incorporated into clinical guidelines and is widely used by the majority of
microbiologic laboratories (13). The Platelia GM assay is an enzyme-linked
immunoassay that can be performed on both serum and BAL. The serum and BAL
GM were approved by the FDA and are used in clinical practice for the evaluation of
patients with possible or probable IA (9). False positive results have been reported

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with the use of antibacterial agents such as piperacillin/tazobactam (14), although this
association is now under debate (15).

According to two large meta-analyses, the sensitivity and specificity of GM on serum

is estimated to be 79% and 81-86%, respectively, when using 0.5 optical density
index (OD) as the cutoff for positivity (12, 16). However, as expected, the sensitivity
decreases and specificity increases when higher OD cut-offs are used (16). These
studies found significant heterogeneity in the results that may have confounded the
performance estimates. Importantly, some of the included studies in both meta-
analyses used at least two positive GM test results to define test positivity, which may
have underestimated the sensitivity and overestimated the specificity of the test in the
pooled analysis. Indeed, in the study by Leeflang et al., when a single sample was
used to define test positivity, sensitivity was 94% and specificity was 61% for the 0.5
OD cut-off. Another notable point to consider is that antifungal prophylaxis was
found to reduce the specificity, while antifungal therapy reduced the sensitivity of the
test (12). Therefore, depending on the context and way of use, the test performance
varies.

GM on BAL is associated with higher accuracy. Indeed, sensitivity and specificity of

the test was found to be 90% and 94% respectively in a large meta-analysis (17).
However, again this result varied according to the cut-off used for GM positivity with
an OD of 1 achieving the best performance with pooled sensitivity and specificity of
85% and 94% respectively in an earlier meta-analysis (17), while an OD of 1.5
showed impressive sensitivity and specificity of 92% and 98% in a more recent
systematic review (18). The disagreement over the ideal cut-off for the test is reflected
on the latest EORTC/MSG guidelines, which avoid recommending a specific cut-off
(9). Interestingly, the high performance of BAL GM was challenged by the largest
cohort trial to-date testing the diagnostic accuracy of GM (19). This study showed
disappointing sensitivity and specificity of 50% and 73%, respectively with a cut-off
of 0.5 OD. These results could be put into context when we consider that all of the
proven and probable IA patients in the cohort were receiving mold-active antifungal
agents at the time the BAL was performed. Indeed, antifungal agents had been shown
to reduce the performance estimates of BAL GM in prior studies (20). However, the
difference in the BAL GM performance estimates among different studies might also
be a reflection of the wide inconsistencies in the diagnostic yield of BAL in general

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(21). In an effort to decrease these inconsistencies, Sampsonas et al. recently
suggested the use of a standardized protocol for BAL in cancer patients (22).

Beta-D-Glucan

Beta-D-Glucan (BDG) is found on the cell wall of most fungal species, with the
exception of Zygomyces and Cryptococcus spp. (23). A test that utilizes a reaction
between free circulating BDG and a zymogen and induces a coagulation cascade has
been used to detect BDG on the serum and plasma of patients with invasive fungal
infections (24). In individual studies, the sensitivity and specificity of the test for the
diagnosis of IA has ranged from 55-95% and 75-96%, respectively (25). In a recent
meta-analysis, the pooled sensitivity and specificity for BDG among patients with
invasive fungal infections was 75.6% and 85.3% respectively and was not
significantly different in the subgroup of studies that assessed the performance of the
test in IA (25). False-positive results are more common in patients on dialysis or those
who are infected with certain Gram-negative bacteria, such as Pseudomonas
aeruginosa (26). The test has been incorporated in the guidelines as a criterion for a
probable invasive fungal infection (9), but one of its main drawbacks is that it cannot
differentiate between Aspergillus spp. and other fungi.

Lateral flow devices

A lateral flow device (LFD) that uses a mouse monoclonal antibody, known as JF5, to
detect a glycoprotein antigen found in the serum and BAL of patients with IA has
recently emerged and holds promise to be used as rapid point-of-care test for IA (27).
An early clinical trial showed that it has comparable performance parameters to those
of GM on serum with a sensitivity and specificity of 81.8% and 98%, respectively,
but has the additional advantages that it is easy to use, requires no technical expertise
for its performance and has a very quick turnaround of results (28). Similarly, a small
pilot study showed that the LFD device can also be used in BAL with a sensitivity and
specificity of 100% and 81% respectively, but false positive findings can occur in
patients who are colonized or infected with other fungi, like Penicillium spp., so
positive results should be interpreted with caution (29). Furthermore, in a recent
prospective cohort BAL LFD for IA demonstrated sensitivity and specificity of 77%
and 92% respectively (30). Despite the promising initial findings, larger prospective
trials are needed before we can reach safe conclusions on the performance of

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Aspergillus spp. LFD as an alternative to BAL GM. One such trial is currently
underway and could pave the way to wider availability of LFD by clinical laboratories
(clinicaltrials.gov identifier NCT02058316).

Volatile compounds

It has been relatively recently discovered that in infected individuals, Aspergillus spp.
tend to produce certain organic compounds that are subsequently exhaled and can be
detected in the breath. These compounds, known as volatile organic compounds
(VOCs), have the potential to easily and rapidly identify infected individuals. Several
reports of detection of 2-pentylfuran on the breath of patients infected with IA have
been published (31, 32). A recent proof-of-principle study used the technology of
“electronic noses” to detect the characteristic VOC signature of Aspergillus spp. and
was able to provide a 100% sensitivity and 83.3% specificity for IA in a high-risk
group of patients (33). Using a slightly more complicated technology with thermal
desorption-gas chromatography/mass spectrometry, Koo et al. were able to report
94% sensitivity and 93% specificity for the detection of IA in 64 prospectively
enrolled high-risk patients (34). These results are indeed promising and have been
embraced with enthusiasm by the infectious disease community (35), but larger
studies are warranted to have a better understanding of the performance of the test in
different populations and the possibility of interactions with antifungal agents or other
medications. For example, a recent in vitro study showed that bisphosphonates, like
alendronate can drastically alter the composition of VOCs produced by A. fumigatus
(36).

Polymerase Chain Reaction

Although polymerase chain reaction (PCR) for the diagnosis of IA has been available
for more than 20 years (37), it has yet to find its place in clinical practice. The main
reason behind this is the lack of standardization with different investigators not being
able to agree to specific guidelines for PCR methodology and interpretation. As a
result, sensitivity and specificity of Aspergillus spp. PCR varies widely from 43-100%
and 64-100%, respectively, on individual studies for PCR performed on serum and
whole blood and 33-100% and 70-100%, respectively, for PCR performed on BAL
(13). Two comprehensive meta-analyses on PCR performed on blood specimens
showed a pooled sensitivity and specificity 84-88% and 75-76%, respectively, thus

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concluding that PCR is unable on its own to confirm or exclude IA (38, 39). Not
surprisingly though, both studies found evidence for significant heterogeneity.
Individual clinical trials evaluating Aspergillus spp. PCR differ on the specimen tested
(serum vs. whole blood), number of positive specimens needed to define test
positivity (one vs. at least two), type of primers used (rRNA vs. mitochondrial vs.
others), and methodology for DNA extraction from clinical specimens.

To eliminate methodological parameters as factors for heterogeneity and allow for

improved interlaboratory comparisons, a multicenter effort was recently completed by
the European Aspergillus PCR Initiative (EAPCRI) to provide guidelines for PCR
methodology. In two multicenter studies, the investigators tested in vitro several
parameters in the methodology of DNA extraction and amplification from whole
blood (40) and serum (41) specimens and issued specific recommendations that
improve the in vitro performance of Aspergillus spp. PCR. These recommendations
are increasingly used by researchers in the field and have been proven to also improve
clinical performance of PCR in high risk patients in a large systematic review (38).
This is a ground-breaking effort, as it opens the door to an environment where PCR
methodologies that are slightly different could become acceptable assays provided
that they meet certain criteria.

The decision on whether to rely on one positive or at least two positive PCR results to
define test positivity depends mostly on the purpose of use of the PCR test. It comes
as no surprise that requiring at least two positive results for test positivity increases
the specificity and decreases the sensitivity of the test. Indeed, in the most recent
meta-analysis at least two positive PCR results demonstrated a sensitivity and
specificity of 64% and 95% respectively, compared to a sensitivity and specificity of
84% and 76% respectively for one positive test result (38). Therefore, if the test is to
be used as a screening method, the single positive approach would be preferable, as in
screening settings it is paramount to have high sensitivity to avoid missing individuals
who might be infected. On the other hand, when using the test as a confirmatory tool,
if for example the test was to be incorporated in the guidelines as a microbiological
criterion to confirm IA, the approach with the highest specificity should be preferred.

Being outside the scope of the EAPCRI effort, the question of whether serum or
whole blood should be used preferentially for PCR testing remains unanswered. Two

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relatively small clinical trials that compared serum and whole blood PCR in the same
patient population failed to show significant differences between the two (42, 43).
Similar results were found by a recent meta-analysis on the subject (38). Nevertheless,
the possibility remains that the power of these studies might not have been high
enough to detect a small but significant difference. In the absence of any evidence to
the contrary though, serum and whole blood PCR should be considered to have
similar performance. In that case, serum PCR might be used preferentially as serum is
easier to process and allows for other serologic tests to be performed at the same time.
BAL can also be used to detect the DNA of Aspergillus spp. PCR performed on BAL
has a pooled sensitivity and specificity of >90% in two recent meta-analyses (44, 45).
However, the sensitivity of the test is reduced significantly down to 77% when
considering only the studies that used the EORTC/MSG criteria as a reference
standard (45). Nevertheless, the high specificity of the test makes it a useful tool for
confirming an infection in a patient with high pre-test probability for the disease.

Screening for IA

As noted in detail above, diagnosis of IA is particularly challenging, perhaps more so

than most other human infections. Because this disease is also associated with a very
high mortality, especially when treatment is delayed, it is not uncommon for patients
at high risk to be treated empirically with mold-active antifungal agents in an effort to
battle early infections and prevent the morbidity and mortality associated with their
dissemination. However, this choice does not come without its drawbacks. One of the
main disadvantages of this approach is that it inevitably leads to the development of
antifungal agent-resistant strains of Aspergillus. Indeed, the emergence of triazole-
resistant A. fumigatus is becoming an increasingly common occurrence in Europe and
other countries and constitutes a threat to public health (46). Also, antifungal agents
are associated with sometimes severe side-effects (47), which may be a necessary evil
in the case of infected individuals, but could have been avoided in people who
received antifungal agents unnecessarily as an empiric measure. Therefore,
identification and validation of a reliable and accurate screening algorithm for IA in
high-risk populations is essential. To that end, two recent randomized controlled trials
evaluating GM and PCR as non-invasive methods for IA screening in patients with
hematologic malignancies were recently published (48, 49). The first study showed
that GM and PCR screening algorithms can reduce the use of empiric antifungal

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agents without increasing mortality from IA (48). Subsequently, other investigators
assessed whether GM alone vs. combined PCR and GM screening is better and found
that the latter is true (49). These are indeed important trials that highlight the efficacy
of GM and PCR as screening modalities. However, we still do not know what the best
screening algorithm is, with different studies following different screening
approaches. Also, despite the fact that these studies do not show any mortality cost in
using GM and PCR based screening to decide on the use of antifungal agents, we
should note that mortality was a secondary outcome in both studies and they might
not have been powered enough to show small differences. In the absence of any
reliable sensitivity and specificity estimates of different GM and PCR based screening
algorithms, more studies will be needed to delineate and validate the screening
strategy with the best clinical potential along with its specific risks and benefits.

Conclusions

Timely diagnosis of IA and early initiation of mold-active antifungal agents is

essential to prevent its high morbidity and mortality. To-date, no single diagnostic test
has been proven to be able on its own to confirm or rule-out the disease in the clinical
setting. While this might change in the future with the discovery of novel diagnostic
methodologies (such as VOCs) and the standardization and improvement of molecular
and serologic tests, for the time being the focus of further clinical trials should be the
creation and validation of diagnostic algorithms that incorporate and take advantage
of multiple available diagnostic tests in the screening and diagnosis of IA.

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References

1. Herbrecht R, Letscher-Bru V, Oprea C, Lioure B, Waller J, Campos F, et al.

Aspergillus galactomannan detection in the diagnosis of invasive aspergillosis in
cancer patients. J Clin Oncol. 2002;20(7):1898-1906.
2. Horvath JA, Dummer S. The use of respiratory-tract cultures in the diagnosis
of invasive pulmonary aspergillosis. Am J Med. 1996;100(2):171-178.
3. Perfect JR, Cox GM, Lee JY, Kauffman CA, de Repentigny L, Chapman SW,
et al. The impact of culture isolation of Aspergillus species: a hospital-based survey of
aspergillosis. Clin Infect Dis. 2001;33(11):1824-1833.
4. Zmeili OS, Soubani AO. Pulmonary aspergillosis: a clinical update. QJM.
2007;100(6):317-334.
5. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr
KA, et al. Treatment of aspergillosis: clinical practice guidelines of the Infectious
Diseases Society of America. Clin Infect Dis. 2008;46(3):327-360.
6. Hot A, Maunoury C, Poiree S, Lanternier F, Viard JP, Loulergue P, et al.
Diagnostic contribution of positron emission tomography with
[18F]fluorodeoxyglucose for invasive fungal infections. Clin Microbiol Infect.
2011;17(3):409-417.
7. Kim JY, Yoo JW, Oh M, Park SH, Shim TS, Choi YY, et al. (18)F-fluoro-2-
deoxy-D-glucose positron emission tomography/computed tomography findings are
different between invasive and noninvasive pulmonary aspergillosis. J Comput Assist
Tomogr. 2013;37(4):596-601.
8. Sharma P, Mukherjee A, Karunanithi S, Bal C, Kumar R. Potential role of
18F-FDG PET/CT in patients with fungal infections. AJR Am J Roentgenol.
2014;203(1):180-189.
9. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et
al. Revised definitions of invasive fungal disease from the European Organization for
Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and
the National Institute of Allergy and Infectious Diseases Mycoses Study Group
(EORTC/MSG) Consensus Group. Clin Infect Dis. 2008;46(12):1813-1821.
10. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, et al.
Defining opportunistic invasive fungal infections in immunocompromised patients
with cancer and hematopoietic stem cell transplants: an international consensus. Clin
Infect Dis. 2002;34(1):7-14.
11. Borlenghi E, Cattaneo C, Capucci MA, Pan A, Quaresmini G, Franco F, et al.
Usefulness of the MSG/IFICG/EORTC diagnostic criteria of invasive pulmonary
aspergillosis in the clinical management of patients with acute leukaemia developing
pulmonary infiltrates. Ann Hematol. 2007;86(3):205-210.
12. Leeflang MM, Debets-Ossenkopp YJ, Visser CE, Scholten RJ, Hooft L,
Bijlmer HA, et al. Galactomannan detection for invasive aspergillosis in
immunocompromized patients. Cochrane Database Syst Rev. 2008(4):CD007394.
13. Arvanitis M, Anagnostou T, Fuchs BB, Caliendo AM, Mylonakis E.
Molecular and nonmolecular diagnostic methods for invasive fungal infections. Clin
Microbiol Rev. 2014;27(3):490-526.
14. Viscoli C, Machetti M, Cappellano P, Bucci B, Bruzzi P, Van Lint MT, et al.
False-positive galactomannan platelia Aspergillus test results for patients receiving
piperacillin-tazobactam. Clin Infect Dis. 2004;38(6):913-916.

This article is protected by copyright. All rights reserved.

15. Mikulska M, Furfaro E, Del Bono V, Raiola AM, Ratto S, Bacigalupo A, et al.
Piperacillin/tazobactam (Tazocin) seems to be no longer responsible for false-positive
results of the galactomannan assay. J Antimicrob Chemother. 2012;67(7):1746-1748.
16. Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis using a
galactomannan assay: a meta-analysis. Clin Infect Dis. 2006;42(10):1417-1427.
17. Guo YL, Chen YQ, Wang K, Qin SM, Wu C, Kong JL. Accuracy of BAL
galactomannan in diagnosing invasive aspergillosis: a bivariate metaanalysis and
systematic review. Chest. 2010;138(4):817-824.
18. Heng SC, Morrissey O, Chen SC, Thursky K, Manser RL, Nation RL, et al.
Utility of bronchoalveolar lavage fluid galactomannan alone or in combination with
PCR for the diagnosis of invasive aspergillosis in adult hematology patients: A
systematic review and meta-analysis. Crit Rev Microbiol. 2013.
19. Affolter K, Tamm M, Jahn K, Halter J, Passweg J, Hirsch HH, et al.
Galactomannan in bronchoalveolar lavage for diagnosing invasive fungal disease. Am
J Respir Crit Care Med. 2014;190(3):309-317.
20. Marr KA, Laverdiere M, Gugel A, Leisenring W. Antifungal therapy
decreases sensitivity of the Aspergillus galactomannan enzyme immunoassay. Clin
Infect Dis. 2005;40(12):1762-1769.
21. Peikert T, Rana S, Edell ES. Safety, diagnostic yield, and therapeutic
implications of flexible bronchoscopy in patients with febrile neutropenia and
pulmonary infiltrates. Mayo Clin Proc. 2005;80(11):1414-1420.
22. Sampsonas F, Kontoyiannis DP, Dickey BF, Evans SE. Performance of a
standardized bronchoalveolar lavage protocol in a comprehensive cancer center: a
prospective 2-year study. Cancer. 2011;117(15):3424-3433.
23. Marty FM, Koo S. Role of (1-->3)-beta-D-glucan in the diagnosis of invasive
aspergillosis. Med Mycol. 2009;47 Suppl 1:S233-240.
24. Hope WW, Walsh TJ, Denning DW. Laboratory diagnosis of invasive
aspergillosis. Lancet Infect Dis. 2005;5(10):609-622.
25. Karageorgopoulos DE, Vouloumanou EK, Ntziora F, Michalopoulos A,
Rafailidis PI, Falagas ME. beta-D-glucan assay for the diagnosis of invasive fungal
infections: a meta-analysis. Clin Infect Dis. 2011;52(6):750-770.
26. Mennink-Kersten MA, Ruegebrink D, Verweij PE. Pseudomonas aeruginosa
as a cause of 1,3-beta-D-glucan assay reactivity. Clin Infect Dis. 2008;46(12):1930-
1931.
27. Thornton CR. Development of an immunochromatographic lateral-flow device
for rapid serodiagnosis of invasive aspergillosis. Clin Vaccine Immunol.
2008;15(7):1095-1105.
28. White PL, Parr C, Thornton C, Barnes RA. Evaluation of real-time PCR,
galactomannan enzyme-linked immunosorbent assay (ELISA), and a novel lateral-
flow device for diagnosis of invasive aspergillosis. J Clin Microbiol.
2013;51(5):1510-1516.
29. Hoenigl M, Koidl C, Duettmann W, Seeber K, Wagner J, Buzina W, et al.
Bronchoalveolar lavage lateral-flow device test for invasive pulmonary aspergillosis
diagnosis in haematological malignancy and solid organ transplant patients. J Infect.
2012;65(6):588-591.
30. Prattes J, Flick H, Pruller F, Koidl C, Raggam RB, Palfner M, et al. Novel
tests for diagnosis of invasive aspergillosis in patients with underlying respiratory
diseases. Am J Respir Crit Care Med. 2014;190(8):922-929.

This article is protected by copyright. All rights reserved.

31. Syhre M, Scotter JM, Chambers ST. Investigation into the production of 2-
Pentylfuran by Aspergillus fumigatus and other respiratory pathogens in vitro and
human breath samples. Med Mycol. 2008;46(3):209-215.
32. Chambers ST, Syhre M, Murdoch DR, McCartin F, Epton MJ. Detection of 2-
pentylfuran in the breath of patients with Aspergillus fumigatus. Med Mycol.
2009;47(5):468-476.
33. de Heer K, van der Schee MP, Zwinderman K, van den Berk IA, Visser CE,
van Oers R, et al. Electronic nose technology for detection of invasive pulmonary
aspergillosis in prolonged chemotherapy-induced neutropenia: a proof-of-principle
study. J Clin Microbiol. 2013;51(5):1490-1495.
34. Koo S, Thomas HR, Daniels SD, Lynch RC, Fortier SM, Shea MM, et al. A
breath fungal secondary metabolite signature to diagnose invasive aspergillosis. Clin
Infect Dis. 2014;59(12):1733-1740.
35. Kontoyiannis DP, Patterson TF. Diagnosis and treatment of invasive fungal
infections in the cancer patient: recent progress and ongoing questions. Clin Infect
Dis. 2014;59 Suppl 5:S356-359.
36. Heddergott C, Calvo AM, Latge JP. The volatome of Aspergillus fumigatus.
Eukaryot Cell. 2014;13(8):1014-1025.
37. Yamakami Y, Hashimoto A, Tokimatsu I, Nasu M. PCR detection of DNA
specific for Aspergillus species in serum of patients with invasive aspergillosis. J Clin
Microbiol. 1996;34(10):2464-2468.
38. Arvanitis M, Ziakas PD, Zacharioudakis IM, Zervou FN, Caliendo AM,
Mylonakis E. PCR in diagnosis of invasive aspergillosis: a meta-analysis of
diagnostic performance. J Clin Microbiol. 2014;52(10):3731-3742.
39. Mengoli C, Cruciani M, Barnes RA, Loeffler J, Donnelly JP. Use of PCR for
diagnosis of invasive aspergillosis: systematic review and meta-analysis. Lancet
Infect Dis. 2009;9(2):89-96.
40. White PL, Bretagne S, Klingspor L, Melchers WJ, McCulloch E, Schulz B, et
al. Aspergillus PCR: one step closer to standardization. J Clin Microbiol.
2010;48(4):1231-1240.
41. White PL, Mengoli C, Bretagne S, Cuenca-Estrella M, Finnstrom N,
Klingspor L, et al. Evaluation of Aspergillus PCR protocols for testing serum
specimens. J Clin Microbiol. 2011;49(11):3842-3848.
42. Springer J, Morton CO, Perry M, Heinz WJ, Paholcsek M, Alzheimer M, et al.
Multicenter comparison of serum and whole-blood specimens for detection of
Aspergillus DNA in high-risk hematological patients. J Clin Microbiol.
2013;51(5):1445-1450.
43. Bernal-Martinez L, Gago S, Buitrago MJ, Gomez-Lopez A, Rodriguez-Tudela
JL, Cuenca-Estrella M. Analysis of performance of a PCR-based assay to detect DNA
of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical
samples. J Clin Microbiol. 2011;49(10):3596-3599.
44. Sun W, Wang K, Gao W, Su X, Qian Q, Lu X, et al. Evaluation of PCR on
bronchoalveolar lavage fluid for diagnosis of invasive aspergillosis: a bivariate
metaanalysis and systematic review. PLoS One. 2011;6(12):e28467.
45. Avni T, Levy I, Sprecher H, Yahav D, Leibovici L, Paul M. Diagnostic
accuracy of PCR alone compared to galactomannan in bronchoalveolar lavage fluid
for diagnosis of invasive pulmonary aspergillosis: a systematic review. J Clin
Microbiol. 2012;50(11):3652-3658.

This article is protected by copyright. All rights reserved.

46. Pham CD, Reiss E, Hagen F, Meis JF, Lockhart SR. Passive surveillance for
azole-resistant Aspergillus fumigatus, United States, 2011-2013. Emerg Infect Dis.
2014;20(9):1498-1503.
47. Powers JH. Issues in clinical trials of prophylaxis of fungal infections. Clin
Infect Dis. 2004;39 Suppl 4:S211-217.
48. Morrissey CO, Chen SC, Sorrell TC, Milliken S, Bardy PG, Bradstock KF, et
al. Galactomannan and PCR versus culture and histology for directing use of
antifungal treatment for invasive aspergillosis in high-risk haematology patients: a
randomised controlled trial. Lancet Infect Dis. 2013;13(6):519-528.
49. Aguado JM, Vazquez L, Fernandez-Ruiz M, Villaescusa T, Ruiz-Camps I,
Barba P, et al. Serum Galactomannan Versus a Combination of Galactomannan and
Polymerase Chain Reaction-Based Aspergillus DNA Detection for Early Therapy of
Invasive Aspergillosis in High-Risk Hematological Patients: A Randomized
Controlled Trial. Clin Infect Dis. 2014.

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Test Specimen Sensitivity Specificity Challenges References

Galactomannan Serum 79%* 81-86%* Decreased [12, 16,

performance in 20]
patients receiving
antifungal agents.

Optimal cut-off
unclear.

Galactomannan BAL 90%* 94%* Optimal cut-off [17-19]

unclear.

Performance in high-
risk patients
challenged by recent
study

Beta glucan Serum 76%* 85%* Non-specific for [25, 26]

Aspergillus spp.

False positives occur

with gram negative
bacteria.

PCR Serum and 84-88%* 75-76%* Non-standardized [38, 39]

whole methodology.
blood

PCR BAL 90-91%* 92-96%* Non-standardized [44, 45]

methodology.

Decreased
performance in studies
that use the
EORTC/MSG criteria
as reference standard.

Lateral flow Serum or 82% on 98% on Performance estimates [28-30]

device BAL serum, 77- serum, 81- derived from single
100% on 92% on small studies. Not
BAL BAL extensively validated
in clinical trials

Volatile organic Exhaled air 94-100% 83-93% Performance estimates [33, 34]
compound derived from few
detection small studies. Not
extensively validated
in clinical trials

BAL: Bronchoalveolar lavage; PCR: Polymerase chain reaction

*Sensitivity and specificity estimates derived from one or more meta-analyses

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