Journal of the Pediatric Infectious Diseases Society

SUPPLEMENT ARTICLE

Role of Molecular Biomarkers in the Diagnosis of Invasive

Fungal Diseases in Children
Anna R. Huppler,1 Brian T. Fisher,2 Thomas Lehrnbecher,3 Thomas J. Walsh,4,5 and William J. Steinbach6,7
1
Department of Pediatrics, Division of Infectious Disease, Medical College of Wisconsin, Children’s Hospital and Health System, Children’s Research Institute, Milwaukee;
2
Division of Pediatric Infectious Diseases, Children’s Hospital of Philadelphia, Pennsylvania; 3Division of Pediatric Hematology and Oncology, Hospital for Children and
Adolescents, Johann Wolfgang Goethe University, Frankfurt, Germany; 4Division of Infectious Diseases, Department of Medicine, Transplantation-Oncology Infectious Diseases
Program, and 5Department of Pediatrics, Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York; and 6Division of Pediatric Infectious Diseases and
7
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina

Invasive fungal diseases are important clinical problems that are often complicated by severe illness and therefore the inability to
use invasive measures to definitively diagnose the disease. Tests for a range of fungal biomarkers that do not require an invasive
sample-collection procedure have been incorporated into adult clinical practice, but pediatric data and pediatric-specific recommen-
dations for some of these diagnostic tools are lacking. In this review, we summarize the published literature and contemporary strat-
egies for using the biomarkers galactomannan, (1→3)-β-d-glucan, Candida mannan antigen and anti-mannan antibody, and fungal
polymerase chain reaction for diagnosing invasive fungal disease in children. Data on biomarker use in neonates and children with
cancer, history of hematopoietic stem cell transplant, or primary immunodeficiency are included. Fungal biomarker tests performed
on blood, other body fluids, or tissue specimens represent promising adjuncts to the diagnostic armamentarium in populations with
a high prevalence of invasive fungal disease, but substantial gaps exist in the correct use and interpretation of these diagnostic tools
in pediatric patients.
Keywords.  β-D-glucan; galactomannan; invasive fungal disease; mannan; polymerase chain reaction.

Invasive fungal diseases (IFDs) are significant causes of mor-
bidity and death in pediatric patients with a compromised
immune system. Definitively diagnosing infection with yeast or
a filamentous fungus is difficult and often requires an invasive
procedure to obtain a standard culture. Most clinical studies
use a composite IFD outcome inclusive of “proven” or “prob-
able” IFD, separating them from “possible” IFD. According to
consensus international guidelines, the distinguishing factor
between a possible or probable IFD designation is the presence
of either direct (eg, microbiologic recovery) or indirect (eg, pos-
itive biomarker result) mycologic criteria [1]. Although species
identification and the availability of susceptibility testing on
culture-positive specimens is a distinct diagnostic advantage for
the treatment team, the opportunity for microbiologic recovery
is not always clinically feasible or timely.
Biomarkers, measured in blood or another clinical spec-
imen, are promising adjuncts to traditional pathologic and
microbiolic tools for diagnosing IFD. Currently, assays to
measure fungal biomarkers, including galactomannan (GM),
Correspondence: A.  R. Huppler, MD, Pediatric Infectious Disease, Children’s Hospital of
Wisconsin, Suite C450, P.O. Box 1997, Milwaukee, WI 53201-1997 (ahuppler@mcw.edu).
Journal of the Pediatric Infectious Diseases Society   2017;6(S1):S32–44
© The Author 2017. Published by Oxford University Press on behalf of The Journal of the
Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/jpids/pix054
(1→3)-β-d-glucan (BDG), Candida mannan antigen and
anti-mannan antibody, and fungal polymerase chain reaction
(PCR) are available to aid in the diagnosis of IFD. A large
number of cohort studies have documented the utility of
these diagnostic tools in adult patients [2–6]. However, with
exception of the GM assay, relatively few data on the use of
these molecular tools in children exist. It is important to note
that the available adult biomarker data cannot be extrapo-
lated seamlessly to pediatric patients for a variety of reasons,
including, but not limited to, differences in the pathophysiol-
ogy of IFDs, the incidence of IFDs, and cutoff values for pos-
itive results in children versus adults. Two English-language
guidelines have been published [7, 8], and they provided spe-
cific guidance for using fungal biomarkers for the diagnosis of
IFDs in children; however, these guidelines address only chil-
dren with cancer or pediatric hematopoietic stem cell trans-
plant (HSCT) recipients.
To interpret the studies included in this review, it is import-
ant to understand the methodologic challenges inherent in bio-
marker studies. All studies on fungal biomarkers are limited by
the lack of a true gold standard for the declaration of an IFD,
which makes it difficult to accurately classify study subjects as
having or not having an IFD. As a surrogate for a gold stan-
dard, the European Organization for Research and Treatment of
Cancer/Invasive Fungal Infections Cooperative Group and the
National Institute of Allergy and Infectious Diseases Mycoses

S32  • JPIDS 2017:6 (Suppl 1) •  Huppler et al

Study Group (EORTC/MSG) published consensus definitions
for IFD in 2002 and revised them in 2008 [1, 9]. Although the
EORTC/MSG criteria have established a systematic approach to
defining IFDs, limitations still exist. A positive GM assay result
is one of the microbiologic criteria that can contribute to the
assignment of probable IFD. Reports of studies that evaluate
the performance characteristics of the GM assay often do not
state how the authors handle the intrinsic bias of using GM
assay results to define a case of IFD. Furthermore, defining the
exact time of onset for an IFD presents difficulties, which leads
to ambiguity when associating a positive fungal biomarker with
an IFD diagnosis. Some studies have interpreted positive bio-
marker test results before a patient has met established EORTC/
MSG criteria for IFD as a true-positive result and applauded
the finding as an improvement on the time to diagnosis of IFD.
However, some positive measurements of biomarker levels in
advance of the patient meeting EORTC/MSG criteria can rep-
resent a false-positive result.
In this review, we highlight current knowledge of the uses
and limitations of fungal biomarkers in the care of neonates
and pediatric patients with cancer, history of HSCT, or primary
immunodeficiency, who are at risk for IFD.
GM ASSAY
The Platelia Aspergillus enzyme-linked GM immunoassay (Bio-
Rad, Hercules, CA) was designed specifically for the early diag-
nosis of invasive aspergillosis (IA) (Table  1). This assay is the
oldest of the major fungal biomarker tests and has been avail-
able in Europe since 1997. It was approved by the US Food and
Drug Administration in 2003 for use in adult patients and in
2006 for use in pediatric patients. The assay uses a sandwich
enzyme-linked immunosorbent assay technique with a rat
anti-GM monoclonal antibody (EB-A2) that recognizes galac-
tofuranose epitopes of the GM molecule as both a capture and
detector antibody. The recommended threshold for a positive
result from a serum specimen is an optical density index of 0.5
or higher. This threshold is supported by the current interna-
tional guidelines for defining IFDs [1].
Adult Data
Before and since its approval, numerous studies that examined
the diagnostic utility of the GM assay in individual adult sub-
populations at high risk for IA across various clinical scenarios
Table 1.  Summary of Fungi Detected by Fungal Biomarkers
Fungal Biomarker Fungal Species Detected
Galactomannan Aspergillus, Penicillium, Paracoccidioides, Histoplasma, Fonsecaea, Cryptococcus
(1→3)-β-d-glucan Aspergillus spp, Candida spp, Fusarium spp, Trichosporon spp, Saccharomyces cerevisiae,
Acremonium spp, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii,
Pneumocystis jirovecii
Candida mannan C albicans, C glabrata, C tropicalis
have been published. A meta-analysis of studies in which serial
surveillance GM testing was used in high-risk populations found
the GM assay to have an overall sensitivity of 71% and specificity
of 89% [2]. Sensitivity was highest in patients with a hemato-
logic malignancy at risk for neutropenia (70%) and for patients
undergoing HSCT (82%), although data on neutrophil counts
were not included. The GM assay had poor sensitivity (22%) in
solid organ transplant recipients [10], likely related to the vari-
ance in pathogenesis in these patients, in whom neutropenia
predisposing to fungal angioinvasion is less common [11]. The
operating characteristics of the assay are also poor in the setting
of mold-active antifungal therapy [12–14]. As with any diagnos-
tic tool, the prevalence of IA in the studied population will affect
the positive predictive value (PPV) and negative predictive value
(NPV) of the GM assay. In the meta-analysis described above,
in the setting of a baseline prevalence (pretest probability) of
5%, the GM assay would have a PPV (posttest probability) of
31% [2]. The PPV increased to 69% with a baseline prevalence of
20%. These findings underscore the importance of understand-
ing the baseline risk for IA in the patient to be tested.
Pediatric Data
To date, 23 studies with the GM assay have been performed in
children, including 18 prospective [15–32] (Table 2) and 5 ret-
rospective [33–37] studies. Many of these studies have been
reviewed [38–40]. The authors of systematic reviews concluded
that the cutoff values and assay operating characteristics in chil-
dren and adults seem similar [38]. In general, specificity of the
GM assay has been good in prospective pediatric studies; most
studies reported a specificity of >87% [39]. Sensitivity was as
high as 100% for proven and/or probable IA, but only in studies
with multiple cases of IA [39], which indicates that GM assay use
should be limited to pediatric populations with a high likelihood
of IA. The GM assay has been used as a screening and a diagnos-
tic tool in studies focused on pediatric patients with cancer or
HSCT recipients; sensitivity and specificity were highly variable,
but the NPVs were consistently high [40]. Important to note is
that it seems that age and weight are not specific factors in GM
assay performance in children; however, false positivity in pre-
mature infants has been reported (discussed in detail later) [41].
As noted previously for adults, the GM assay might be less
sensitive in certain pediatric patients who do not have neutro-
penia as their primary risk factor for IA. A study that included
16 children with IA and a primary immunodeficiency (chronic
Fungal Species Not Detected
Saccharomyces, Candida, Pichia
Cryptococcus spp, Blastomyces dermatitidis (in yeast form), Absidia
spp, Mucor spp, Rhizopus spp; potentially lower detection of C
parapsilosis and C famata
C parapsilosis, other fungi
Role of Fungal Biomarkers in Children  • JPIDS 2017:6 (Suppl 1) • S33
 Table 2.  Prospective Pediatric GM Assay Studies

No. of GM Proven/ False-Positive Rate

Authors Subjects Sample Years No. of Children Samples ODI Cutoff IFD Definition Indication for Testing Probable IA Sensitivity (%) Specificity (%) (%)a
Rohrlich et al, 1996 [15] Children NR 37 425 ≥0.93 Guiot et al (1994) [115] Surveillance (neutropenia) 10 100 92.6 5.7
Sulahian et al, 2001 [16] Adults and 1995–1998 347 2376 ≥1.5 Locally defined Surveillance (neutropenia) 9 100 89.9 10.1
children
Herbrecht et al, 2002 [17] Adults and 1997–2001 42 3294 ≥1.5 EORTC/MSG 2002 Diagnosis (F&N or suspected pul- 2b NR 47.6 44 (F&N), 75
children monary infection) or surveillance (HSCT)c
(HSCT)

S34  • JPIDS 2017:6 (Suppl 1) •  Huppler et al

Challier et al, 2004 [18] Adults and 1999–2001 20 207d ≥1.0 EORTC/MSG 2002 Clinically diagnosed IA 12b NR NR NR
children
El-Mahallawy et al, 2006 [19] Children 2003–2004 91 NR ≥0.5 EORTC/MSG 2002 Diagnosis (F&N) 28 79 61 NR
Hovi et al, 2007 [20] Children 2000–2002 98 932 ≥0.5 EORTC/MSG 2002 Surveillance (induction chemother- 1 NR 93 5.7
apy for ALL, chemotherapy for
AML, HSCT with neutropenia or
immunosuppression for GVHD)
Steinbach et al, 2007 [21] Children 2003–2004 64 826 ≥0.5 EORTC/MSG 2002 Surveillance (HSCT with neutropenia 1 0 98.4e 1.6
or GVHD)
Hayden et al, 2008 [22] Children NR 56 990 ≥0.5 EORTC/MSG 2002 Surveillance (neutropenia or immu- 17 65.7 87.2 1.0
nosuppressive risk)
Armenian et al, 2009 [23] Children 2006–2007 68 1086 ≥0.5 EORTC/MSG 2002 Surveillance (neutropenia, steroid 3 NR 98.7 0.1
use or HSCT)
Zhang et al, 2009 [24] Children NR 88 NR ≥0.5 NR NR 14b 71.4 91.9 NR
Fisher et al, 2012 [25] Children 2004–2007 198 1865 ≥0.5 EORTC/MSG 2002 Surveillance (neutropenia or HSCT) 1 0 95 5.2
Badiee et al, 2012 [26] Children 2008–2009 62 230 ≥0.5 EORTC/MSG 2008 Surveillance (hematology disorders) 10 90 92 NR
Choi et al, 2013 [27] Children 2007–2010 83 640 ≥0.5 EORTC/MSG 2008 Surveillance (F&N, GVHD, cortico- 23 91.3 81.7 18.3
steroids or HSCT) and diagnosis
Jha et al, 2013 [29] Children 2010–2011 78 NR ≥0.5 EORTC/MSG 2002 Diagnosis (fever) 25b 84 38 NR
Dinand et al, 2016 [28] Children 2007–2011 145 405 ≥0.7 EORTC/MSG 2008 Diagnosis (F&N) 45b 82.2 82.5 13.7
Gefen et al, 2015 [30] Children 2010–2011 46 510 ≥0.5 NR Surveillance (HSCT or high-risk NR 80 66 NR
leukemia)
Gupta et al, 2017 [31] Children 2013–2014 125 125 ≥0.5 EORTC/MSG 2008 Diagnosis (fever or F&N) 62 81.9 49 NR
Loeffler et al, 2017 [32] Children 2012–2015 39 543 ≥0.5 EORTC/MSG 2008 Surveillance (HSCT) 4 100 NR 2.1

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; EORTC/MSG, 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; F&N, fever and
neutropenia; GM, galactomannan; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplant; IA, invasive aspergillosis; IFD, invasive fungal disease; NR, not reported; ODI, optical density index.
a
According to sample, not patient, unless noted.
b
Includes possible cases that constitute the majority of cases.
c
False-positive rate according to patient, not sample.
d
Pooled samples from adults and children.
e
Excluded patients treated with piperacillin-tazobactam.
granulomatous disease [CGD] [n = 10] or hyperimmunoglob-
ulin E syndrome [Job syndrome] [n = 6]) found that GM was
detected in the serum of only 25% of the patients with IA and
CGD or hyperimmunoglobulin E syndrome versus 80% (24
of 30) in children with IA and neutropenia (P = .0004) [42].
In addition, the authors of recent reviews highlighted the high
false-negativity rate of the GM assay in the setting of patients
with CGD and an IFD in general and in those with Aspergillus
nidulans infection in particular [43, 44].
In 2 published guidelines, pediatric data were considered,
and recommendations for GM testing in children were formu-
lated. The first publication is the pediatric-specific European
guidelines for the diagnosis, prevention, and treatment of IFDs
in children with cancer or those undergoing HSCT [7]. The
authors noted that the GM assay results in operating characteris-
tics in children similar to those in adult patients and that the use
of GM for monitoring in children is warranted and reasonable
[7]. Monitoring was defined as serial screening of serum GM
level twice per week in children at high risk for IFD. A specific
note was made that performance of the GM assay with serum
from pediatric patients under nonsurveillance conditions (ie,
evaluation of a pulmonary infiltrate) is unclear. Weaker sup-
port exists for use of the GM assay on bronchoalveolar lavage
(BAL) specimens or cerebrospinal fluid (CSF). The second pub-
lication is the pediatric-specific international fever and neutro-
penia guideline, originally published in 2012 and updated in
2017 [8, 45]. The original guideline supported GM surveillance
testing in hospitalized neutropenic patients at high risk (weak
recommendation, moderate-quality evidence) [45]. The 2017
update no longer contains specific recommendations for GM
surveillance testing. Regarding the use of fungal biomarker test-
ing to guide empirical antifungal management in patients with
febrile neutropenia that persists despite antibacterial therapy,
the guidelines recommend consideration of not routinely test-
ing serum GM levels (weak recommendation, moderate quality
evidence) [8]. The rationale for this recommendation includes
concerns for the low PPV in many studies and the fact that the
biomarker is limited to detection of Aspergillus species and thus
would not identify other fungal pathogens that could present in
children with prolonged fever and neutropenia. In addition, the
consistently high NPV for Aspergillus might not be as clinically
informative in this setting when other mold pathogens could
be the cause of the prolonged fever. Authors of the guidelines
expressed concern about the utility of GM testing in children
who are receiving mold-active antifungal prophylaxis [7] or
did not comment because of the lack of data [8]. Mold-active
antifungal prophylaxis is not recommended universally for chil-
dren at risk for IA, but some guidelines support prophylaxis in
centers with a high baseline incidence of invasive mold infec-
tion [7, 46]. Centers that use such prophylaxis need to consider
whether surveillance GM testing is less effective and might opt
for symptom-directed testing, as recommended in a recent
adult-cohort publication [13]. The prevailing data indicate that
mold-active antifungal prophylaxis nullifies the signal of serum
GM such that surveillance is not recommended (strong recom-
mendation, high-quality evidence) by the current Infectious
Diseases Society of America aspergillosis treatment guidelines
for patients who are receiving mold-active antifungal prophy-
laxis [47]. However, it might still be useful to apply the GM
assay to bronchoscopy specimens from patients on antimold
prophylaxis who are undergoing bronchoscopy for an infec-
tious concern. Future results from pediatric-specific compara-
tive effectiveness studies on the utility of mold-active antifungal
prophylaxis and GM surveillance testing in this setting might
help clarify ideal approaches for pediatric clinicians.
Most studies to date have focused on assessing the GM assay
on serum specimens; however, the assay has been performed
on other specimens, including BAL fluid, urine, and CSF. Two
studies retrospectively examined GM levels in BAL specimens
from pediatric patients [35, 36]. Each study found excellent cor-
relation between serum and BAL GM levels, validating findings
in adult patients. The authors of 1 study suggested an optimal
pediatric optical density index cutoff of 0.87 in BAL fluid [35],
similar to the 1.0 cutoff proposed for adult patients [48] and
the 0.85 cutoff suggested by receiver operator curve analyses
in adult patients [49]. A recent prospective pediatric case-con-
trol study used a cutoff of 0.5 for GM in BAL fluid, similar to
that used for serum, resulting in a sensitivity of 87.5% and a
PPV of 93.33% [50]. Several adult studies found that the GM
assay on BAL fluid is more sensitive than the serum GM assay
[49, 51], and the results of 3 pediatric studies suggested that
the combined use of both modalities led to improved overall
detection. Another pediatric study evaluated surveillance urine
and blood GM testing in patients with prolonged neutropenia.
Sensitivity of the GM assay on urine specimens was high, but a
large number of false-positive results occurred when standard
thresholds were used [25]. The measurement of GM levels in
CSF was reported in several case reports and case series, includ-
ing 3 pediatric case reports [52–54].
False-Positive GM Assay Results
Early reports on pediatric GM testing suggested an unaccept-
ably high false-positive rate for children compared to that for
adults. One study included surveillance testing results in chil-
dren with a hematologic malignancy and adult patients who
were undergoing allogeneic HSCT [16]. The reported false-pos-
itive rates were 10.1% (34 of 338) in children and only 2.5% (10
of 406) in an adult cohort. The medical records of 25 pediatric
patients were reviewed, and the investigators concluded that the
presence of concurrent mucositis, which might the favor pas-
sage of GM from the digestive tract, or early antifungal therapy,
which might diminish the detectability of IFD, could have led to
false-positive reactions. In another large study of adult and pedi-
atric patients with fever of unknown origin [17], false-positive
Role of Fungal Biomarkers in Children  • JPIDS 2017:6 (Suppl 1) • S35
results again were observed in children significantly more fre-
quently (44.0%) than in adults (0.9%) (P < .0001). In addition,
3 of the 11 children with false-positive results had repeated
false-positive results, whereas the adult patients had only 1
false-positive result. A more recent retrospective study of 141
children with acute lymphoblastic leukemia who were screened
twice per week during periods of neutropenia reported that
more than half (52.1%) of the 179 positive serum samples were
false positives and that one-fourth (26.1%) were of undeter-
mined significance [37].
False-positive results are of particular concern in premature
neonates. In 1 study, 6 premature infants without documented
IA had a positive GM assay result in surveillance testing. In
repeat testing, 5 of these premature infants were found to test
positive persistently [41]. The etiology of these false-positive
results is hypothesized to result from cross-reactivity of a mem-
brane-associated molecule of Bifidobacterium bifidum subsp
pennsylvanicum, which was found to mimic the epitope rec-
ognized by the EB-A2 antibody used in the GM-detection kit
[55]. Bifidobacterium spp comprise 91% and 75% of the total
microflora in breastfed and formula-fed neonates, respectively.
Various brands of milk formula have been reported to yield pos-
itive GM assay results, with speculated translocation into serum
samples after ingestion, but 3 samples of human milk that were
tested in that study had a negative result [56].
In early reports, some batches of piperacillin-tazobactam
were found to yield false-positive reactions in the GM assay
[57]. Several older studies excluded patients who were receiving
piperacillin-tazobactam or automatically classified GM results
for such patients as false-positive. It is important to note that in
recent years, manufacturing changes for piperacillin-tazobac-
tam have eliminated cross-reactivity with the GM assay [58].
BDG ASSAY
BDG is an integral cell wall glucose polysaccharide found in
the majority of fungi. It is notable that this polysaccharide is
also the target of echinocandin antifungal agents. The BDG
assay can detect Aspergillus spp, Candida spp, Fusarium spp,
Trichosporon spp, Saccharomyces cerevisiae, Acremonium spp,
Coccidioides immitis, Histoplasma capsulatum, Sporothrix
schenckii, and Pneumocystis jirovecii (Table 1). Also notable is
that several clinically important fungi, including Cryptococcus
spp, Blastomyces dermatitidis (in yeast form), Absidia spp,
Mucor spp, and Rhizopus spp, are not detected by the BDG assay
because of their low or absent BDG production. Factor G is a
coagulation factor of the horseshoe crab that serves as a highly
sensitive natural detector of BDG via a modified Limulus endo-
toxin assay. Factor G is prepared from amebocytes of either the
North American horseshoe crab (Limulus polyphemus) or the
Japanese horseshoe crab (Tachypleus tridentatus), and assay
properties differ in chromogenic reactivities and cutoff values
S36  • JPIDS 2017:6 (Suppl 1) •  Huppler et al
depending on the source of crab species. Each of the 5 commer-
cially available assays relies on lysate from a specific horseshoe
crab species. Fungitell (Associates of Cape Cod, East Falmouth,
Massachusetts), which uses the North American horseshoe
crab, is approved and available in the United States and Europe.
Another assay that uses the North American horseshoe crab is
available for research use only (Glucatell [Associates of Cape
Cod]). Fungitec G (Seikagaku, Tokyo), Wako (Wako, Japan),
and Maruha (Maruha-Nichiro, Tokyo) are available only in
Japan and use the Japanese horseshoe crab as the source for the
assay lysate. The Fungitell BDG assay was approved by the US
Food and Drug Administration in 2004.
Adult Data
Numerous adult trials that investigated the utility of the BDG
assay have been completed. A meta-analysis of studies that
investigated the utility of BDG testing for detecting IFDs in
adult patients with hematologic or oncologic disease deter-
mined that the sensitivity, specificity, PPV, and NPV of 2 con-
secutive tests were 49.6%, 98.9%, 83.5%, and 94.6%, respectively
[3]. An IFD prevalence of 10% was used to estimate the PPV and
NPV. The majority of the 6 studies included in the meta-anal-
ysis used a screening methodology, whereas 1 study measured
BDG for suspected IFD. BDG has also been investigated in the
context of Pneumocystis jirovecii pneumonia (PCP) diagnosis.
In 2 meta-analyses, which mostly included adult human immu-
nodeficiency virus–positive patients, serum BDG levels had a
sensitivity of 94.8% to 96% for PCP diagnosed by the presence
of the pathogen in sputum or BAL fluid [59, 60].
Pediatric Data
There have been few publications on the utility of BDG test-
ing to detect IFDs in children at risk. The studies that have
been published have been limited primarily to case reports or
small series [61–69]. Five prospective studies with the BDG
assay were performed in children with a hematologic malig-
nancy or pediatric HSCT recipients [26, 31, 70–72] (Table 3).
Sensitivities of the BDG assay ranged from 50% to 82%, and
specificities ranged from 46% to 82%. In 1 recent prospective
study of pediatric patients (between birth and 21 years of age)
with fungemia or microbiologically proven invasive candidiasis,
the specificity increased from 67% to 76% and the NPV from
91.7% to 92.6% with an increase in the cutoff from 80 to 100 pg/
mL [72]. Although specific numbers were not provided, 4 of the
5 studies noted high rates of false-positive results. The studies
also were limited in some cases by use of a noncommercial assay
[70] or definitions of IFDs that were not always consistent with
EORTC/MSG criteria [71].
A recent retrospective study of 118 children, which included
790 serum samples, examined the BDG assay with a cutoff of
≥80 pg/mL using the Fungitell assay as surveillance for IFD in
patients with neutropenia [73]. Using EORTC/MSG criteria to
 diagnose IFD, 9 unique proven or probable cases were found,

False-Positive

Abbreviations: BDG, (1→3)-β-d-glucan; EORTC/MSG, 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; F&N, fever and neutropenia; HSCT, hematopoietic stem cell
and the sensitivity and specificity of the BDG assay were 75%

Rate

NR

NR

NR
NR

NR
and 56%, respectively. Specificity increased to 90% when 2 posi-
tive test results were required, but then the sensitivity decreased
to 50%. Exclusion of patients on antifungal therapy also
improved the performance of assays for BDG [73]. A case-con-
Specificity

trol study using a novel BDG-detection assay, the GKT assay

47.2
(%)
82.4

46

67
76
82
(Gold Mountain Tech Development Co., Ltd, Beijing) included
56 patients with documented candidemia and 210 patients with-
out candidemia [74]. The patients with candidemia included 38
Sensitivity

80.2 who were born prematurely, 10 with a gastrointestinal abnor-

(%)
81.8

mality, 6 with congenital heart disease, and 3 with a neurologic

50

77
78
80

abnormality. The sensitivity and specificity for candidemia were

68% and 91%, respectively; it was noted, however, that the BDG
levels were low or negative in patients with Candida parapsilo-
Probable
Proven/

IA

10

62

27
22

sis or Candida famata candidemia, which might be a result of

smaller amounts of BDG in the cell walls of these species.
Diagnosis (persistent

tologic disorders)

The authors of 1 pediatric case series reported detectable

Surveillance (hema-

Surveillance (HSCT)

positive cultures
Diagnosis (fever or
Indication for

Surveillance (all

CSF BDG levels in 9 patients with probable or proven central

and control
population)
Testing

nervous system Candida or Aspergillus infection [75]. BDG

fever)

F&N)

results reverted to negative in every patient who completed

treatment. A recent detailed study of successful treatment of
Aspergillus ventriculitis in a pediatric patient revealed the diag-
EORTC/MSG 2008c
EORTC/MSG 2008

EORTC/MSG 2008

EORTC/MSG 2008
IFD Definition

nostic value of CSF BDG testing and the pivotal role for mon-
NR

itoring therapeutic response [54]. CSF GM testing lacked the

analytical dynamic range to be used for monitoring therapeutic
response in this case. Very few data on the use of BDG testing
BDG Cutoff (pg/mL)

for the diagnosis of PCP in pediatric patients are available. One

small case series found BDG levels of >500 pg/dL in 3 pediatric
≥100
≥10a

≥80

≥80
≥80
≥80

patients with confirmed or presumed PCP [76].

It is important to note that the appropriate cutoff for a posi-
tive BDG assay result in children has not been established, and
data suggest that the adult cutoff of 80 pg/mL with the Fungitell
Samples
No. of
BDG

or Glucatell assay might not be appropriate for children. The

230

702
125

NR
NR

differences were first noted in the initial pediatric BDG study

that retrospectively examined BDG levels in the serum of chil-
dren without an IFD. BDG baseline values in this setting were
Children
No. of

127
62

125
130

34b

approximately one-third higher in children than in adults [77].

transplant; IA, invasive aspergillosis; IFD, invasive fungal disease; NR, not reported.

Similar concerns about increased baseline values were raised in a

Table 3.  Prospective Pediatric BDG Assay Studies

retrospective study of 61 neonates with or without invasive can-

didiasis. The optimal cutoff for distinguishing candidiasis with
2012–2014

2012–2015
2008–2009

2013–2014
2007–2008
Sample
Years

the Fungitell assay was 125 pg/mL [78]. Additional research

studies with large pediatric cohorts of children at risk for IFDs
are needed to better define the optimal threshold for positivity
Systemic mold prophylaxis routinely administered.

in children. Because of the limited data on this assay in children,

Subjects

Children

Children
Children

Children
Children

there are no definitive recommendations in any pediatric or neo-

Inconsistent application of criteria.

natal guidelines for its routine use. The European pediatric-spe-

cific guideline for the diagnosis, prevention, and treatment of
Badiee et al, 2012 [26]

Koltze et al, 2015 [71]

Gupta et al, 2016 [31]

Salvatore et al, 2016

Zhao et al, 2009 [70]

Noncommercial assay.

IFDs in children with cancer or those undergoing HSCT notes

insufficient data to base clinical decisions on BDG assay results
Authors

[7]. The pediatric-specific international fever and neutropenia

[72]

guideline recommends not using the BDG assay to guide empiric

a

Role of Fungal Biomarkers in Children  • JPIDS 2017:6 (Suppl 1) • S37

antifungal management in patients with fever and neutropenia
that persist while the patient is undergoing antibacterial therapy
(strong recommendations, low-quality evidence) [8].
Potential causes of false-positive results with the assay
include blood products, hemodialysis, surgical gauze, pipera-
cillin-tazobactam, ampicillin-clavulanate, mucositis, alcohol
swabs, replacement immunoglobulin, and others (reviewed
in reference 79). Persistently positive BDG levels also were
reported after treatment of candidemia in a pediatric HSCT
recipient, and exhaustive studies revealed no persistent focus of
infection [67]. A recent study in pediatric intensive care unit
patients found no difference in BDG levels in children with and
those without bacteremia [80]. False-negative BDG assay results
can occur in patients who are already undergoing antifungal
therapy. High concentrations of bilirubin and triglycerides
also reportedly inhibit the performance of the assay and cause
false-negative BDG values [81].
FUNGAL PCR
Multiple publications have reported on the utility of nucleic
acid–based detection of fungal DNA through PCR. These
studies have focused most commonly on Aspergillus spp or
Candida spp as the pathogens of interest. Quantitative real-time
PCR testing of BAL fluid or induced sputum is widely used to
diagnose PCP. Various PCR techniques were used in the pub-
lished studies, including pathogen-specific PCR assays (for
the detection of a single genus or species) or “panfungal” PCR
approaches that rely on conserved gene sequences located in
ribosomal RNA genes or the spacer DNA between genes (inter-
nal transcribed spacer [ITS]). Multiple gene targets and primers
have been used for both the pathogen-specific and panfungal
PCRs. In addition to the variability in the gene targets, there are
many variations in protocols for sample preparation, specimen
type, and reaction format. The specimen type can be plasma,
serum, or whole blood. Studies have used reaction formats of
standard PCR, nested PCR, semi-nested PCR, multiplex PCR,
and real-time PCR techniques, each of which carries differ-
ent advantages and challenges. The European Aspergillus PCR
Initiative (EAPCRI) was established to address these issues and
optimize protocols and accelerate routine clinical use for labo-
ratory diagnosis of aspergillosis [82].
Adult Data
A meta-analysis summarized 25 studies that evaluated blood-
based Aspergillus PCR in patients with a hematologic disease
who were at high risk and reported a pooled sensitivity of 84%
and a specificity of 76%. When the definition of true positive
required 2 positive PCR results, the specificity increased to
94% [4]. For PCR diagnosis of invasive candidiasis, a separate
meta-analysis reviewed 54 studies (unspecified age in 22 stud-
ies, adults alone in 17, children alone in 8, and both children
S38  • JPIDS 2017:6 (Suppl 1) •  Huppler et al
and adults in 7) that included 4694 patients. The study designs
varied in testing methodology and patient inclusions; both
neutropenic and nonneutropenic patients were included, and
disease was categorized as candidemia or invasive candidiasis
(proven, probable, or possible) according to EORTC/MSG 2008
criteria. When used in the context of suspected invasive can-
didiasis, PCR had a sensitivity of 95% and specificity of 92%
for diagnosing candidemia. When it was used to study patients
with proven or probable invasive candidiasis versus true-nega-
tive patients, sensitivity and specificity were similar (93% and
95%, respectively) [5].
Recent guidelines for the diagnosis of PCP in patients with
a hematologic malignancy and HSCT recipients recommended
real-time PCR for the routine diagnosis of PCP [83]. BAL fluid
is the preferred specimen, because a negative PCR result from
a specimen collected noninvasively is not considered adequate
to rule out PCP.
After the development of DNA-extraction methods, prim-
ers, and probes for quantitative PCR (qPCR) assays of mucor-
mycosis in BAL fluid and plasma [84], several studies revealed
the value of qPCR in the laboratory diagnosis of pulmonary and
disseminated mucormycosis in predominantly adult popula-
tions of immunocompromised patients and patients with burn
wounds [85–87]. Circulating DNA from Mucorales spp was
detected in 9 of 10 patients between 68 and 3 days before the
diagnosis of mucormycosis was confirmed by positive culture
or histopathologic examination. However, the qPCR assay was
negative in a patient with disseminated mucormycosis caused
by a Lichtheimia sp [85].
A recent advance in PCR technology for the diagnosis
of candidemia is the T2Candida assay (T2Biosystems, Inc,
Lexington, Massachusetts) [88]. After a DNA-amplification
step, T2-weighted magnetic resonance is used to detect the
amplified product within 3 to 5 hours. The assay is performed
on whole blood and can detect 5 distinct Candida species:
Candida albicans, Candida tropicalis, Candida parapsilosis,
Candida krusei, and Candida glabrata. In a study that compared
the T2Candida assay to standard blood cultures, the sensitivity
and specificity for detection of the 5 species from seeded blood
samples were 100% and 97.8%, respectively [88]. A second
study used patient-derived blood culture specimens manually
inoculated with Candida to evaluate the T2Candida assay. The
overall sensitivity and specificity for 250 contrived samples were
91.1% and 99.4%, respectively [89].
Pediatric Data
Eleven prospective studies of Aspergillus or pan-fungal serum
PCR in children have been performed [18, 19, 23, 26, 31, 32,
90–94] (Table 4), and 2 retrospective studies of Aspergillus
or panfungal PCR have been performed in children [95, 96].
Results in children have been mixed, with sensitivities rang-
ing from 63% to 100% depending on the study, specific patient
 Table 4.  Prospective Pediatric Aspergillus or Panfungal PCR Studies

Sample No. of No. of PCR Indication for Proven/

Authors Subjects Years Children Type of PCR Samples IFD Definition Testing Probable IA Sensitivity (%) Specificity (%) False-Positive Rate (%)
Bialek et al, 2002 [92] Children NR 17 Aspergillus 71 EORTC/MSG 2002 Surveillance (before 3a NR NR NR
and after HSCT)
Challier et al, 2004 [18] Adults and 1999–2001 20 Aspergillus 28S rRNA 207b EORTC/MSG 2002 Clinically diagnosed IA 12a NR NR NR
children
El-Mahallawy et al, 2006 [19] Children 2003–2004 91 Panfungal 18S rRNA NR EORTC/MSG 2002 Diagnosis (F&N) 28 75 92 8.3
Cesaro et al, 2008 [93] Children 2004–2005 62 Aspergillus 18S rRNA 536 EORTC/MSG 2002 Diagnosis (F&N, HSCT 8 63 81 19c
with fever, or sus-
pected IA)
Armenian et al, 2009 [23] Children 2006–2007 52 Aspergillus 28S rRNA 554 EORTC/MSG 2002 Surveillance (neutro- 3 NR NR 87.5
penia, steroid use,
or HSCT)
Hummel et al, 2009 [90] Children 2000–2007 71 Aspergillus 18S rRNA 291d EORTC/MSG 2002 Diagnosis 5 80 81 NR
Mandhaniya et al, 2012 [94] Children 2008–2009 29 Aspergillus and 29 EORTC/MSG 2002 Diagnosis (fever 0e 0 36 NR
Candida 28S rRNA while on mold
prophylaxis)
Badiee et al, 2012 [26] Children 2008–2009 62 Aspergillus 230 EORTC/MSG 2008 Surveillance (hemato- 10 80 96.2 NR
logic disorders)
Reinwald et al, 2014 [91] Children 1997–2000 95 Aspergillus 967 EORTC/MSG 2008 Diagnosis (fever) 0 NR NR 10
Gupta et al, 2016 [31] Children 2013–2014 125 Panfungal 28S rRNA 125 EORTC/MSG 2008 Diagnosis (fever, F&N) 62 82.7 54 NR
Loeffler et al, 2017 [32] Children 2012–2015 39 Aspergillus ITS1- 543 EORTC/MSG 2008 Surveillance (HSCT) 4 100 NR 3.9
5.8S rRNA

Abbreviations: EORTC/MSG, 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; F&N, fever and neutropenia; HSCT, hematopoietic stem cell transplantation; IA, invasive
aspergillosis; NR, not reported; PCR, polymerase chain reaction; rRNA, ribosomal RNA.
a
Includes possible cases.
b
Pooled samples from adults and children.
c
False-positive rate according to patient, not sample.
d
Nonblood samples included.
e
Patient with mucormycosis not included.

Role of Fungal Biomarkers in Children  • JPIDS 2017:6 (Suppl 1) • S39

population, and selection of PCR assay. A recent retrospective
study examined the combined utility of a multifungal DNA
microarray and Aspergillus-specific PCR assay for the detection
of IFD in biopsy specimens from 18 proven and 29 probable
cases of IFD, 36 of which represented proven or probable IA
[96]. The combined assays had a sensitivity of 79% and specific-
ity of 69% for any IFD, including IA. When evaluated on blood
samples from the same patients, the sensitivity of the PCRs was
reduced to 33%, and specificity decreased 75%. Three studies
were performed to examine Candida PCR in children [62, 65,
97]. The largest study included 54 neonates and children with a
central venous catheter and persistent fever [97]. Blood cultures
from 8 patients were positive for Candida spp. Candida DNA
via multiplex nested PCR targeting the ITS region was detected
in 13 patients, including the 8 children with a positive blood
culture result, and an additional 5 patients who tested positive
on the basis of PCR results only. PCR results were negative in
all 28 control group patients who had no evidence of any type
of bloodstream infection. The time to results was only 24 hours,
which is an improvement on standard culture methods. In addi-
tion to potential delayed time to identification, blood cultures
can also “miss” 50% of invasive candidiasis cases [79]. A second
study found positive results for Candida PCR in 5 cases of “can-
didiasis” among pediatric surgery patients [62]. A limitation of
that study was using a definition of candidiasis based on positive
fungal biomarkers in 3 of the 5 cases, including 1 that was pos-
itive only by Candida PCR. The third study focused on patients
colonized with Candida spp and found that all Candida serum
sample PCR results were negative in 20 colonized patients [65].
One small pediatric study evaluated the T2Candida assay with
whole blood from 15 children with candidemia and 9 children
without candidemia [98]. The concordance of these results with
blood culture results was 100% despite low sample volumes that
required reducing T2Candida assay testing volumes from the
standard of 4 to 2 mL.
Two pediatric studies of PCP PCR testing of upper and
lower respiratory tract specimens in children at risk for PCP
have been performed [99, 100]. PCR results were positive in
100% of the cases with positive direct immunofluorescence
for the cystic forms of P. jirovecii. Samuel et al [99] found that
nasopharyngeal aspirates in children with PCP had similar
rates of PCR reactivity when compared to BAL fluid for the
detection of PCP. However, it was noted that PCR results were
7-fold more likely to be positive than those of immunofluo-
rescence, and the specificity of a positive PCR test for true
disease could not be determined. A later study from the same
center also found that PCR was approximately 2.5 times more
sensitive in the detection of PCP in BAL fluid than other
methods [100].
There is a paucity of reported pediatric cases with mucormy-
cosis diagnosed with qPCR [101, 102]. In 1 case, a 3-year-old
child who underwent allogeneic HSCT and had disseminated
S40  • JPIDS 2017:6 (Suppl 1) •  Huppler et al
mucormycosis caused by Rhizomucor pusillus was serially mon-
itored with serum qPCR [101].
PCR technology for the diagnosis of IFD potentially has sev-
eral advantages, including theoretically higher sensitivity than
culture- or antigen-based methodologies. However, the lack of
a standardized methodology limits widespread uptake of PCR
diagnostics for detecting Candida and Aspergillus infections.
False-positive results caused by nonspecific primers or the
presence of clinically irrelevant fungal DNA are also a concern.
Until we have standardized methods and acceptable operating
characteristics for a standardized approach, use of PCR for the
detection of IFD in children cannot be routinely recommended.
The pediatric-specific European guidelines for the diagnosis,
prevention, and treatment of IFDs in children with cancer or
those undergoing HSCT make no general recommendation for
fungal PCR use [7]. The pediatric-specific international fever
and neutropenia guideline recommends against the routine
use of fungal PCR testing of blood to guide empiric antifungal
management in patients with fever and neutropenia that persist
while the patient is undergoing antibacterial therapy (strong
recommendations, moderate-quality evidence) [8].
CANDIDA MANNAN ANTIGEN AND ANTI-MANNAN
ANTIBODY
Cell wall mannan comprises approximately 7% of Candida
cell dry weight and is a major circulating antigen during infec-
tion. The assay was derived originally against C albicans man-
nan using the EB-CA1 monoclonal antibody [103, 104]. The
Candida mannan antigen and anti-mannan antibody assays are
available currently in Europe (Bio-Rad Platelia Candida antigen
and Platelia Candida antibody) but are not commercially avail-
able in the United States.
Adult Data
A review of 14 studies on adult patients with invasive candi-
diasis examined the performance of Candida mannan antigen
and anti-mannan antibody in patients with a hematologic dis-
ease and in ICU patients. The tests performed best in combi-
nation; used together, they had a sensitivity of 83% (increased
from approximately 60% for the individual assays) and a spec-
ificity of 86% [6]. Sensitivity seems to be best for C albicans,
followed by C glabrata and C tropicalis, most likely because of
the similarity between the mannan antigens of these species. In
a subset of studies on patients with a hematologic malignancy,
sensitivities ranged from 71% to 100% and specificities from
53% to 92% [6]. A recent prospective study of 67 adults with
a hematologic malignancy found a high false-positive rate for
anti-mannan antibody and a combined specificity for mannan
antigen and anti-mannan antibody of 74.8% [105]. In an adult
nonneutropenic ICU population with invasive candidiasis, the
two tests combined had a sensitivity and specificity of 54.8%
and 59.9%, respectively [106]. In a head-to-head comparison,
the sensitivity of mannan antigen and anti-mannan antibody
combined were comparable to that of the BDG assay but with a
lower specificity [103].
Pediatric Data
All of the pediatric Candida antigen reports to date have
included, often exclusively, neonates [61, 69, 107–112]. The
largest study examined 184 preterm infants and required a
mannan level of ≥0.5 ng/mL in at least 2 serum samples to be
considered positive [110]. Only 70 patients with at least 3 man-
nan measurements were included in the final analysis. Twelve
neonates were found to have invasive candidiasis according to
conventional testing; the sensitivity of the assay was 92% (11
of 12). Important to note is that in 8 of the 12 cases of inva-
sive candidiasis, the mannan antigen result was positive before
the blood culture by a median of 8.5 days (range, 4–18 days). A
false-negative assay result occurred in a patient with C parapsi-
losis candidemia. This lack of detection for C parapsilosis is con-
sistent with the difference in sensitivity for antigenic detection
by EB-CA1 monoclonal antibody of the different Candida spe-
cies mannose epitopes. Because the assay was derived against C
albicans mannan, it is less likely to detect C parapsilosis mannan
[103, 104]. The limited ability of the mannan assay to detec-
tion C parapsilosis was confirmed in other studies with a higher
prevalence of C parapsilosis events [61]. All 10 neonates in that
study had a positive BDG assay result, whereas only 70% had
a positive result for mannan antigen. The patients who tested
positive for mannan antigen were diagnosed with non-parapsi-
losis Candida infection. Only limited data on the utility of this
assay for nonserum specimens exist. The detection of mannan
antigen in BAL fluid from preterm neonates has been studied as
an indication for empirical antifungal therapy in infants at high
risk [111, 112], and 1 such report of CSF testing in a neonate
and a child was published [107].
Nonneonatal pediatric data regarding the Candida mannan
and/or anti-mannan assay are lacking. Only 5 nonneonatal
patients with candidemia were included a pediatric study of
Candida mannan [61]. The BDG assay result was positive for
all 5 patients with a hematologic malignancy, whereas Candida
mannan was detected in only 2 of these 5 candidemic patients
(1 with C albicans and 1 with Candida lusitaniae infections). A
pediatric intensive care unit study reported 100% sensitivity of
the mannan antigen assay for candidemia and 60% sensitivity
for the anti-mannan antibody assay [113]. A high false-positive
rate was found (23%), but the authors speculated that many of
these results represented undiagnosed invasive candidiasis as
evidenced by clinical response to antifungal therapy. A study
of pediatric patients with cancer colonized with Candida spp
reported a low rate of positive test results for mannan antigen
(10%) [65]. Even with additional data, the persistent inability of
the mannan antigen assay to detect C parapsilosis is worrisome,
because C parapsilosis is the causative pathogen in a significant
proportion of all pediatric invasive candidiasis [114].
SUMMARY RECOMMENDATIONS FOR FUNGAL
BIOMARKER USE IN PEDIATRIC PATIENTS
The GM assay has operating characteristics in nonneonatal
children similar to those in adults, and the sensitivity and spec-
ificity are optimized for populations of patients with underly-
ing neutropenia. However, the GM assay has a high rate of false
positivity in neonatal patients and poor sensitivity in patients
with a primary immunodeficiency or solid organ transplant
recipients without neutropenia. Use of the BDG assay in neo-
nates and older children has not been well described, and
pediatric guidelines currently discourage reliance on it for diag-
nosis, because the optimal cutoff is not known and infections
with several important fungal pathogens are not detected by the
assay. Fungal PCR testing from blood currently suffers from a
lack of standardization. The Candida mannan antigen/antibody
approach has also undergone only limited testing in nonneona-
tal children, leading to an inability to offer clear recommenda-
tions on its use.
The ultimate use of biomarkers in diagnosing IFDs might
require multiple assays in combination to overcome limits in sen-
sitivity and specificity. Pediatric-specific studies of the biomarkers
alone and combined are needed to define the limitations of each
assay and to design the optimal multipronged diagnostic strategy.
One recent study found improved sensitivity by combining bio-
markers [31]. Furthermore, as with any diagnostic test, the utility
of testing these biomarkers is founded on the pretest probability
of infection in the child to be tested. Even if the sensitivity and
specificity are optimal for a given biomarker or combination of
biomarkers, the results might not be useful if the likelihood of
IFD is low. Therefore, pediatric-specific guidelines on fungal bio-
marker testing must be individualized for the clinical scenario that
incorporates the estimated IFD prevalence (ie, pretest probability)
for that clinical scenario. For instance, biomarker testing might
be useful when a child has a condition that predisposes him or
her to IFD (eg, prolonged neutropenia) and presents with signs
and symptoms of IFD, but testing for that same biomarker might
not be useful for surveillance testing in the absence of signs and
symptoms of IFD. In addition, an understanding of the epidemi-
ology of pathogens that can cause IFD in specific patient popu-
lations is important when testing for specific fungal biomarkers.
For instance, a negative result for a biomarker with specificity to a
fungal pathogen (eg, the GM assay and Aspergillus) might provide
a high NPV for that pathogen but not provide any guidance on the
risk for other potential fungal pathogens. Extrapolating current
pediatric biomarker data to other clinical scenarios is difficult,
such as scenarios with differences in the rates of antimold prophy-
laxis use or specific immunosuppressive agents used. Continual
study and updates to recommendations are necessary.
Role of Fungal Biomarkers in Children  • JPIDS 2017:6 (Suppl 1) • S41

Notes
Financial support.  This work was supported in part by the National
Institutes of Health (grants 1R01 AI03315-A1 and 1R01 HD081044-01 to
B.T.F and W.J.S.), the Henry Schueler Foundation and the Save Our Sick
Children Foundation (to T.J.W.), and the Medical College of Wisconsin
Department of Pediatrics and Children’s Hospital of Wisconsin Research
Institute (to A.R.H.).
Supplement sponsorship.  This article appears as part of the supple-
ment “State of the Art Diagnosis of Pediatric Invasive Fungal Disease:
Recommendations From the Joint European Organization for the Treatment
of Cancer/Mycoses Study Group (EORTC/MSG) Pediatric Committee,”
sponsored by Astellas.
Potential conflicts of interest.  B.T.F. has received research funding from
Ansun, Enzon, and Wyeth and currently receives research funding from
Pfizer and Merck. T.L. is currently receiving a research grant from Gilead
Sciences, is a consultant for Merck/MSD, Basilea, and Gilead Sciences, and
is on the speaker’s bureau for Astellas, Gilead Sciences, Merck/MSD, and
Pfizer. W.J.S. has received basic science research grants from Astellas and is
on advisory boards for Merck, Astellas, and Gilead. T.J.W. receives research
grants for experimental and clinical antimicrobial pharmacotherapeutics
from Astellas, Cubist, Theravance, The Medicines Company, Allergan,
Novartis, Merck, and Pfizer and has served as consultant to Astellas, Actavis,
ContraFect, Drais, iCo, Novartis, Pfizer, Methylgene, SigmaTau, and Trius.
A.R.H.: No reported conflicts. All authors have submitted the ICMJE Form
for Disclosure of Potential Conflicts of Interest. Conflicts that the editors
consider relevant to the content of the manuscript have been disclosed.
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