CE Update

Submitted 1.7.11 | Revision Received 2.24.11 | Accepted 3.3.11

(1-3)-β-D-Glucan Assay: A Review of its Laboratory

and Clinical Application

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William F. Wright, DO, MPH,1 Sue B. Overman, MA, SM(ASCP),2 Julie A. Ribes, MD, PhD2
(1Division of Infectious Diseases, Department of Medicine, 2Clinical Microbiology Laboratory, Department of Pathology and Laboratory
Medicine, University of Kentucky College of Medicine, AB Chandler Medical Center, Lexington, KY)
DOI: 10.1309/LM8BW8QNV7NZBROG

Abstract invasive sample that can be used to aid in Glossary

A new fungal surrogate marker, (1-3)-β-D
glucan, offers a noninvasive method for
the potential surveillance and diagnosis of
invasive fungal infections. Invasive fungal
infections have long been associated with
significantly high morbidity and mortality on
hematology-oncology wards and recipients
of either solid-organ or hematopoietic stem
cell transplantation. The diagnoses of invasive
fungal infections have historically been made
difficult by the need for invasive methods.
(1-3)-β-D-glucan testing requires a minimally
the diagnosis of an invasive fungal infection
as well as monitor the response to treatment.
One disadvantage of (1-3)-β-D-glucan testing
is that a positive test alone lacks sufficient
sensitivity and specificity for a definitive
diagnosis. While formal guidelines for the use
of (1-3)-β-D-glucan testing are lacking, this
chromogenic assay provides a new opportunity
for testing at-risk populations. A review and
recommendation for its laboratory and clinical
application are provided.
(1-3)-β-D-glucan: A polysaccharide
component of the cell wall of most fungi
pyrogen test. An assay used to determine if
a pharmaceutical or medical device intended
for human use will stimulate fever.
Keywords: (1-3)-β-D glucan, beta glucan
assay, invasive fungal infections

After reading this article, readers should be able to discuss what (1-3)- Microbiology 71103 questions and corresponding answer form are
β-D glucans are, how they are detected in clinical biological samples, located after this CE Update on page 686.
the evidence to support laboratory testing and monitoring, the limitations
of testing, and the appropriate clinical setting for testing.

Invasive fungal infections remain a significant problem in
hematopoietic stem cell transplant (HSCT) and solid organ
transplantation (SOT) recipients with changing epidemiologic
patterns during the previous 2 decades.1,2 Despite advances
in technology and therapy, invasive fungal infections are still
associated with significantly high morbidity and mortality.3
Corresponding Author
William F. Wright, DO, MPH
william.wright@uky.edu
Abbreviations
HSCT, hematopoietic stem cell transplant; SOT, solid organ
transplantation; FDA, Food & Drug Administration; LAL, limulus
amebocyte lysate; USP, United States Pharmacopoeia; SST, serum
separator tube; NPV, negative predictive value; PPV, positive pre-
dictive value; PCP, Pneumocystis jirovecii pneumonia; IA, invasive
Aspergillosis; EORTC-IFICG, European Organization for Research
and Treatment of Cancer/Invasive Fungal Infections Cooperative
Group
Among SOT recipients, Candida and Aspergillus pathogens
continue to be most often implicated as the cause of infec-
tions.1 However, Aspergillus species and other filamentous
molds, such as Fusarium, Scedosporium, and the Zygomycetes,
are more commonly associated with invasive fungal infections
in HSCT recipients.2
Measurement of biologic markers, such as the galacto-
mannan Aspergillus antigen and the fungal wall component
(1-3)-β-D-glucan, offers a noninvasive method for the detec-
tion of invasive fungal infections. Historically, the diagnosis
of invasive fungal infections has been made difficult by the
need for tissue biopsies for cultures and histological exami-
nation.4 Furthermore, clinical and radiological findings do
not have sufficient diagnostic sensitivity and specificity to
be helpful. This review will discuss the U.S. Food & Drug
Administration (FDA) approved fungal wall component
(1-3)-β-D-glucan assay and its role as a surrogate marker for
invasive fungal infections.
What Are β-Glucans?
Until recently, the cell wall of fungi has been viewed as
an inert exoskeleton with the primary role to simply provide
structure and support to the organism.5 Based on cumulative

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CE Update
data, the fungal cell wall is now viewed as a dynamic structure
rather than an inert structure. In other words, the fungal cell
wall is continuously undergoing the processes of assembly
and remodeling during cell growth as a result of mechanical
or chemical stresses.6 The dynamic fungal cell wall functions
as a protective barrier, providing structure and stability to the
organism, as well as enabling the organism to penetrate or in-
vade tissues.5,6 The main structural constituents of the fungal
cell wall are polysaccharides.4-6 For the majority of fungi, the
structural core polysaccharides are composed of glucan, chitin,
and mannan.7 Glucan is the most important and abundant
polysaccharide component of the cell wall of most fungi.5
While the cell wall of yeast has been suggested to contain less
glucan than filamentous molds, glucans are a major constitu-
ent of the cell wall of saprophytic and pathogenic fungi with
the exception of Mucor, Rhizopus, Blastomyces dermatitidis,
and Cryptococcus species.4,7-9
The glucan component is predominantly composed of
glucose polymers linked in a linear arrangement by carbons
1 and 3 through a glycosidic bond with a beta configuration
(Figure 1) to form the (1-3)-β-D-glucan backbone.7 The bio-
synthesis of this polysaccharide backbone involves the trans-
portation of glucose subunits to the plasma membrane, where
they are then transported across the plasma membrane and
arranged by a linear β (1-3) glycosidic bond.5 The enzyme
C(6)H2OH
C(5) O
C(4) C(1)
C(3) C(2)
C(6)H2OH
*Glycosidic link
C(5) O test and the new clotting pathway. In 1977, the FDA approved
C(4) C(1)
C(3) C(2)
Figure 1_Schematic of (1-3) Glycosidic Bond.5
(1,3)-β-D-Glucan
Factor G Activated Factor G
Proclotting enzyme Clotting enzyme
Boc-Leu-Gly-Arg-p-nitroanilide p-nitroanilide
(Colorless) (Yellow)
Figure 2_Beta-Glucan Limulus Amebocyte Lysate Reaction.11
680 LABMEDICINE ■ Volume 42 Number 11 ■ November 2011 labmedicine.com
responsible for the formation of this polysaccharide backbone
is (1-3)-β-D-glucan synthase.4-7 The polysaccharide backbone
is typically 1500 glucose subunits in length, but within each
chain branching occurs with either a (1-4) or (1-6) glycosidic
bond.7 The branching assignments are highly variable and
specific to the fungal species.
While incorporated within the fungal cell wall (1-3)-β-
D-glucan typically exists as an insoluble structure. In the
presence of blood or other body fluids, (1-3)-β-D-glucan
transforms into single helix, triple helix (most common), or
random coil forms and are rendered soluble.4,5,7 This soluble
(1-3)-β-D-glucan may be capable of modulating the immune
system by inhibiting leukocyte phagocytosis.10 Details regard-
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ing the release and kinetics of soluble (1-3)-β-D-glucan in the
systemic circulation or body fluids of patients with proven or
probable invasive fungal infections is limited.4
Background of β-Glucan Detection
In 1956, a group from the Johns Hopkins Marine
Biological Laboratory in Massachusetts observed an exten-
sive clotting reaction when endotoxin was injected into the
bloodstream of the North American Horseshoe Crab, Limulus
polyphemus.11 Later it was determined that mobile ameba-like
cells, analogous to phagocytes, within the bloodstream of
Limulus polyphemus contained factor C, which is responsible
for initiating the clotting cascade called the limulus amebocyte
lysate (LAL).11 Factor C, a serine protease zymogen, is obtained
by separating amebocytes from the blue-colored plasma
(hemolymph) followed by suspending the cells in distilled
water, where they are then osmotically lysed.11-13
Prior to the discovery of LAL, approval of pharmaceutical
or medical devices intended for human use required pyrogen
testing using the United States Pharmacopoeia (USP) Pyrogen
Test.11 The solution or device was deemed pyrogenic (fever
causing) and rejected if fever was observed in a rabbit exposed
to these products. Following the discovery of the LAL, many
pharmaceutical solutions or medical devices were tested for
significant bacterial contamination using the classic pyrogen
the use of the LAL as an alternative pyrogen test when
evaluating the approval of intravenous pharmaceuticals or
biological solutions and medical devices (eg, prosthetic heart
valves or prosthetic orthopedic devices).
In 1968, a carboxy-methylated beta-glucan that was
being evaluated as an anti-tumor agent was observed to induce
clotting with the LAL despite the absence of demonstratable
bacterial contamination.12 A second serine protease zymogen,
Factor G, was subsequently determined to be the factor
responsible for initiating the LAL clotting cascade by this
beta-glucan determinant.12,13 The assays developed specifically
to measure fungal beta-glucan typically use serum and rely
on the activation of the LAL clotting cascade as the biological
principle of detection.4,13 The beta-glucan LAL pathway is
summarized in Figure 2.
The following is a general description of the prin-
ciples for the beta-glucan LAL pathway used in the serum
(1-3)-β-D-glucan assay. As mentioned, most of the soluble
beta-glucan typically exist as triple-helix forms and require
conversion to single-strand forms by exposing the serum
sample to an alkaline reagent prior to the beta-glucan LAL
reaction.4 Additionally, the alkaline pre-treatment appears to
 CE Update

reduce the chances of false-positive and false-negative reac-
tions through the inactivation of serine proteases and serine
protease inhibitors, both of which are normally found in
human serum. Following pretreatment with the alkaline
reagent, the beta-glucan LAL reaction is then initiated by the
single-strand beta-glucan binding to the alpha-subunit of fac-
tor G, activating its serine protease subunit.14 Activated factor
G then converts a proclotting enzyme to an activated clotting
enzyme.11,14 This activated clotting enzyme then cleaves a
chromogenic substance, p-nitroanilide, from a synthetic
peptide in the beta-glucan LAL that has replaced the original
clot-forming coagulogen protein. The chromogen, p-nitroan-
ilide, is colorless when attached to a peptide but changes to a
should not be tested due to the inaccurate spectrophotometric
values that can occur with these samples (Table 2).
Two trials compared the performance of the original
(1-3)-β-D-glucan assay, Fungitec-G, with the diagnosis of
invasive fungal infections.15,16 A large multicenter trial con-
ducted by Obayashi and colleagues15 demonstrated that serum
beta-glucan testing is an effective approach to diagnosing inva-
sive fungal infections. Microbiologically, the majority of docu-
mented fungal infections were Candida or Aspergillus species.
In normal volunteers, the plasma concentration of (1-3)-β-D-
glucan was found to be 10 pg/mL. The cut-off value defin-
ing a positive test result was established as 20 pg/mL, using
the Fungitec assay. Of the 179 patients enrolled in the trial,

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yellow color when cleaved from the synthetic peptide.14 While
the original LAL was based on observing the end-point of the
gel clot formation, the beta-glucan LAL is a colorimetric assay
and the end-point is based on determining the absorbance at
450 nm.11,14 Finally, it has been observed that serum samples
have an inherent yellow color that may alter the end-point
absorbance (overestimate); therefore, 1 variation of the beta-
glucan LAL determines the absorbance of the diazo derivative
of p-nitroanilide, which is purple.11
What Are the Available Beta-Glucan Assays?
Currently, 5 beta-glucan assays are available for use (Table
1): Fungitell (Associates of Cape Cod, East Falmouth, MA),
Endosafe-PTS (Charles River Laboratories, Charleston, SC),
Fungitec-G (Seikagaku Biobusiness, Tokyo, Japan), beta-
Glucan Test (Waco Pure Chemical Industries, Osaka, Japan),
and BGSTAR β-Glucan Test (Maruha, Tokyo, Japan). The
Endosafe-PTS and beta-Glucan Test kits are intended only
for research purposes and not for the diagnosis of invasive
fungal infection in clinical samples. Each kit intended for
the in vitro diagnosis of an invasive fungal infection contains
lyophilized horseshoe crab coagulation factor (factor G) and
the chromogenic substrate p-nitroanilide (Boc-Leu-Gly-Arg-
p-nitroanilide). Typically, 3-5 mL of blood is collected into
a serum separator tube (SST) with the minimum amount of
serum volume recommended being 0.5 mL for adults and
0.2 mL for pediatric patients. Following pretreatment of the
sample with an alkaline solution, the reconstituted coagula-
tion factor and chromogenic substrate are combined with
the clinical serum sample and then incubated. Cleavage of
p-nitroanilide from the chromogenic peptide produces the
yellow color that is then measured spectrophotometrically.
The general outline for the assay procedure is summarized in
Figure 3. Blood samples that are lipemic, icteric, or hemolyzed
119 patients had an underlying hematologic malignancy or
hematologic disorder, and 41 of these patients had biopsy-
verified invasive fungal infections. Of these 41 patients, 37
demonstrated serum (1-3)-β-D-glucan concentrations above
the determined cut-off value. Overall, excellent performance was
described with a sensitivity of 76% (31/41), specificity of 100%
(135/161), positive predictive value (PPV) of 59% (37/63),
and a negative predictive value (NPV) of 97% (135/139).
Most of the false-positive results were attributed to concurrent
bacteremia, use of hemodialysis, or treatment involving the use
of human immunoglobulin products.15,16
Investigators in the United States evaluated the perfor-
mance of the beta-glucan assay Glucatell (now known as
Fungitell) as a diagnostic adjunct for invasive fungal infections.17
In a single-center trial conducted by Odabasi and colleagues,17
Glucatell was directly compared to the Fungitec-G assay for
the detection of serum (1-3)-β-D-glucan. Although Glucatell
used reagents from the North American Horseshoe Crab
(Limulus polyphemus) and was reported to be less reactive
than the reagents from the Asian Horseshoe Crab (Tachypleus
tridentatus) used in the Fungitec-G assay, the 2 assays were
equally efficacious in diagnosing invasive fungal infec-
tions. Values for the normal plasma concentration of serum
(1-3)-β-D-glucan and the laboratory cut-off were determined
from 30 healthy adults and 30 candidemic patients. Normal
volunteers had an average of 17 pg/mL of the (1-3)-β-D-
glucan in their samples, while the majority of patients
with demonstrated invasive fungal disease had >60 pg/mL.
Therefore, the definitive positive value was determined to be
>80 pg/mL. Of the 283 neutropenic patients enrolled in the
trial, 16 patients with a proven invasive fungal infection (ie,
biopsy proven) and 4 patients with a possible invasive fungal
infection demonstrated serum (1-3)-β-D-glucan concentra-
tions above the determined cut-off value. The majority of
proven fungal infections were attributed to Candida species.
Overall, the authors described the performance of the assay,

Table 1_Available Beta-D-Glucan Assays

Kit Manufacturer FDA Approved Crab Species Cut-off Value

Fungitell Associates of Cape Cod (U.S.) Yes Limulus polyphemus (colormetric) 60-80 pg/mL
Endosafe-PTS glucan Charles River Laboratories (U.S.) No Limulus polyphemus (colormetric) 10-1000 pg/mL
Fungitec G-MK Seikagaku Biobusiness (Japan) No Tachypleus tridentalus (colormetric) 20 pg/mL
β-glucan test Waco Pure Chemical Industries (Japan) No Tachypleus tridentalus (turbidimetric) 11 pg/mL
BGSTAR β-glucan test Maruha (Japan) No Tachypleus tridentalus (colormetric) 11 pg/mL

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CE Update
20 uL alkaline
pretreatment solution
incubate 10 min at 37°C
Reconstitute Fungitell Lysate
(Factor G and chromogenic peptide)
5 uL serum
Reconstitute Glucan stock
and make standards
Triple helix glucan coverts
to single stranded
Add 100 uL Fungitell Lysate
to each well
Incubate plate at 37°C for 40 minutes,
measuring increase in optical density
at 450 nm – Concentration of Glucan is
calculated against the standard curve
Figure 3_Summary of the beta-glucan assay procedure for Fungitell.4,17
Table 2_Sources of Error for the Beta-Glucan Assay
A. Assay Procedure
1. Mislabeled or unlabeled samples
2. Bacterial or fungal contaminated equipment
3. Non-calibrated spectrophotometer
B. False-positive reactions
1. Concurrent bacteremia (most commonly Streptococcus species)
2. Hemodialysis cellulose membranes and filters
3. Immunoglobulin products (eg, IVIG)
4. Human serum (due to inherent yellow color)
5. Drugs such as Crestin, Lentinan, Schizophyllan, and Scleroglucan
C. False-negative reactions
1. Lipemic blood samples
2. Hemolyzed blood samples
3. Certain fungi that lack significant levels of (1-3)-β-D-glucan such
as Zygomycetes, Cryptococcus neoformans, and Blastomyces
dermatitidis.4,7-9
using 1 serum sample, with a sensitivity of 100%, specificity
of 90%, positive-predictive value of 43%, and a negative-pre-
dictive value of 100%. Optimal sensitivity and specificity was
reached if 2 positive test results on sequential specimens were
used to reflect a true positive test result. Finally, as with the
Fungitec-G assay, most false-positive reactions were attributed
to concurrent bacteremia, use of hemodialysis, or treatment
involving the use of certain human immunoglobulin products.
Since 1976, all new medical devices requiring a premar-
ket approval process are automatically referred to as a class
III medical device (a device for which premarket approval is
required) under the Medical Device Amendment.18 Medical
devices that have completed a premarket approval process and
682 LABMEDICINE ■ Volume 42 Number 11 ■ November 2011 labmedicine.com
are deemed effective and safe
for clinical use are then classi-
fied as a class II medical device
(devices subject to special con-
trols such as performance stan-
dards, post-market surveillance,
patient regulations, or guidance
documents).18 In March 2004,
the FDA filed a petition to au-
tomatically classify Glucatell in
the Federal Registry as a class
III device prior to the premar-
Add 25 uL Standards to
ket approval process.19 Upon
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appropriate wells
review of the clinical trial data,
the FDA reclassified Glucatell
as a class II medical device in
May 2004 for introduction
into interstate commerce for
commercial distribution as a
serological reagent for the de-
tection of (1-3)-β-D-glucan.
Now known as Fungitell, this
is the only FDA-approved de-
vice for the detection of serum
(1-3)-β-D-glucan for use as an
adjunct in the diagnosis of
invasive fungal infections.
Discussion
Within the United States and elsewhere, a few trials have
been conducted to demonstrate the usefulness of beta-glucan
testing with the Fungitell assay in patients with hemato-
logic malignancies at risk for invasive fungal infections.20-24
Unfortunately, comparative data with beta-glucan testing in-
volving patients with solid-organ malignancies or solid-organ
transplantation and invasive fungal infections have not been
described. Data from 4 trials suggest that serum (1-3)-β-D-
glucan testing is an effective pan-fungal marker to aid in the
screening and diagnosis of invasive fungal infections (Table
3).20,22-24 A U.S. trial by Pickering and colleagues23 used
the recommended 80 pg/mL positive cut-off value in serum
samples from 8 different patient groups for the diagnosis of
an invasive fungal infection. They established the 60-79 pg/
mL as the indeterminate range for result reporting, with the
negative range for the assay being below 60 pg/mL. When the
serum samples were compared to samples from healthy blood
donors, the overall sensitivity and specificity of the assay for
the diagnosis of an invasive fungal infection using the 80 pg/
mL cut-off value for positive was 93% and 72%, respectively.
The authors concluded that the assay was primarily useful
in excluding invasive fungal infections due to the high NPV
(97.8%) and relatively high proportion of false-positive re-
sults (10%). False-positive results were attributed to concur-
rent bacteremia (most commonly Streptococcus pneumonia).
False-negative results were attributed to lipemic or hemolyzed
blood samples, as the elevated concentration of bilirubin and
triglycerides are inhibitory to the assay. Ostrosky-Zeichner
and colleagues24 reviewed the literature to determine the op-
timal cut-off values to be used to define patients with positive
results. Comparing the data from several centers, they found
there was a large range of positive cut-off values being used
(range, 40-150 pg/mL). The lower the cut-off value used in
 CE Update

each study, the higher the sensitivity of detecting true positive
patients, but this lowered the overall level of specificity seen
in these studies. Conversely, the higher the cut-off value, the
lower the sensitivity and the higher the specificity of detection
of patients with invasive fungal disease. They concluded that
the optimal sensitivity and specificity for the assay fell between
the 60 pg/mL and 80 pg/mL range that had been defined as
indeterminate and positive test results. Optimal sensitivity
and specificity of detection of invasive fungal infections was
achieved by repeating testing twice each week in the at-risk
patient populations. False-positive results were attributed to
the use of hemodialysis cellulose membranes, hemodialysis
filters, and immunoglobulin products used in this patient
the authors reported a sensitivity and specificity of 87.5%
and 89.6%, respectively, for the diagnosis of IA. Using the
combination of 2 noninvasive tests, the beta-glucan assay and
the Aspergillus galactomannan antigen assay, the trial showed
a sensitivity of 87.5% and specificity of 100% for the diag-
nosis of IA. While false-positive results occurred at a rate of
10.3%, the NPVs remained at 96.3%.20 The authors noted
that the (1-3)-β-D-glucan results were positive earlier than
the galactomannan results, but that all of the patients who
ultimately were found to have IA developed positive results
using both assays.
Results from Persat and colleagues21 suggest that the
Fungitell assay may be useful as a noninvasive test for PCP.

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population. Additionally, some drugs (eg, lentinan, crestin, Of 20 patients with proven PCP, all tested positive using the
scleroglucan, and schizophyllan) used in intensive care units beta-glucan assay, most producing levels >500 pg/mL. The
all contain glucans that may cause false positive test results. authors concluded that beta-glucan testing might be a useful
Results from a prospective trial evaluating the usefulness noninvasive test for the diagnosis of pneumocystosis if these
of measuring serum (1-3)-β-D-glucan concentrations using finding are confirmed in additional patient trials. Finally,
a single serum sample and a determined cut-off value of >60 beta-glucan testing might be useful in the diagnosis
pg/mL as a screening test for the early diagnosis of an invasive of other invasive fungal infections (eg, histoplasmosis) with
fungal infection showed a sensitivity and specificity of 88.9% the exception of Mucor, Rhizopus, Blastomyces dermatitidis,
and 19.8%, respectively.22 If the cut-off value was increased to and Cryptococcus species, but again, more trials are needed to
80 pg/mL, the sensitivity was 88.9% and the specificity was confirm assay performance and to determine the role of this
32.6%. When 2 positive test results on sequential serum samples assay detecting infections with these pathogens.8,9,15-17,20,24
were required for definitive identification of positive patients, When looking at these studies together, optimizing beta-
the sensitivity decreased by almost half while the specific- glucan test sensitivity and specificity depended on testing
ity increased only marginally. Optimal test performance was patients multiple times and using a cut-off value for positive
found using the 80 pg/mL cut-off to define positives, and of 80 pg/mL. The NPV and PPV depended on the prevalence
if no repeat positive test results on sequential specimens of invasive fungal disease in the patient population, which for
was required. Further, the authors of this trial agreed with most of these studies was <10%, accounting for the vast varia-
Pickering and colleagues23 that, due to the excellent NPV of tion in NPV and PPV seen in these studies.
the assay, the Fungitell assay serves best to identify those patients
without invasive fungal infection rather than identifying
those for whom infection has actually been detected. Finally, Therapeutic Monitoring
20
Pazos and colleagues used a cut-off value of 120 pg/mL in Although the surveillance and diagnosis of invasive fungal
40 neutropenic patients using Fungitell as a screening assay infections with beta-glucan testing in high-risk patients seems
with twice-weekly sampling and reported a sensitivity and logical, this surrogate fungal marker may also be useful for
specificity of 87.5% and 89.6%, respectively. Even with this therapeutic monitoring.20,25 This preemptive approach was
exceedingly high cut-off value to define positive patients, these evaluated by Pazos and colleagues20 in 5 neutropenic patients
authors reported a false-positive rate of about 10%. Pazos with a proven diagnosis of IA receiving either amphotericin B
and colleagues20 and others17 had observed that true positives and caspofungin or amphotericin B alone. With twice-weekly
were detected approximately 9-10 days prior
to the onset of clinical manifestations of the
invasive fungal disease.
Although the majority of trials evaluat-
ing the usefulness of beta-glucan testing Table 3_Summary of Trials for the Fungitell Assay for the Diagnosis
involve both Candida and Aspergillus spe- of an Invasive Fungal Infection Using 1 Serum Sample at the Various
cies, 2 trials have compared data for the Cut-off Values
diagnosis of either invasive aspergillosis
Trial Sensitivity Specificity PPV NPV
(IA) or Pneumocystis jirovecii pneumonia
(PCP). 20,21 A European trial by Pazos and Odabasi and colleagues17
colleagues20 prospectively enrolled 40 pa-   (>60 pg/mL) 100% 90.0% 43.0% 100%
tients with hematologic malignancies and Pazos and colleagues20
neutropenia to evaluate the contribution of   (>120 pg/mL) 87.5% 89.6% 70.0% 96.3%
Racil and colleagues22
measuring (1-3)-β-D-glucan concentrations   (>60 pg/mL) 88.9% 19.8% 10.4% 94.4%
to diagnose IA. Of the 40 patients, there Racil and colleagues22
were 5 proven IA, 3 probable IA, and 3 pos-   (>80 pg/mL) 88.9% 32.6% 12.1% 96.6%
sible IA based on the definitions as outlined Ostrosky-Zeichner and colleagues24
  (>60 pg/mL) 69.9% 87.1% 83.8% 75.1%
by the European Organization for Research Ostrosky-Zeichner and colleagues24
and Treatment of Cancer/Invasive Fungal   (>80 pg/mL) 64.4% 92.4% 89.0% 75.1%
Infections Cooperative Group (EORTC- Pickering and colleagues23
IFICG). Using a cut-off value of 120 pg/mL   (>60 pg/mL) 93.3% 77.2% 51.9% 97.8%
for the serum (1-3)-β-D-glucan concentration,

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CE Update
testing, patients who responded to therapy demonstrated a
rapid decline in serum (1-3)-β-D-glucan concentrations by
week 2 and eventually concentrations fell below the determined
positive cut-off value by week 4. In contrast, a continuous in-
crease in serum (1-3)-β-D-glucan concentrations was observed
in those patients not responding to the initiation of antifungal
treatment. Additionally, Kawagishi and colleagues25 observed
a rapid decline in serum (1-3)-β-D-glucan concentrations in a
living-donor liver-transplant recipient receiving antimicrobial
therapy for PCP. While these authors concluded that beta-
glucan testing is useful in predicting therapeutic outcome, the
frequency of monitoring and the role of this surrogate fungal
marker in the management of patients with proven or suspected
invasive fungal infections have not been fully established.20,25
Testing of Specimens Other Than Serum
An additional area of study warranting further investi-
gation is the use of beta-glucan testing on specimens other
than serum. One study published in Japanese26 found high
levels of beta-glucan in the pleural fluid of a patient with IA.
This same study found that only a slight elevation of beta-
glucan was seen in the cerebrospinal fluid from a patient with
cryptococcal meningitis, likely reflecting the small amount
of beta-glucan produced by this organism. The pleural and
cerebrospinal fluids from patients with no fungal infections
demonstrated beta-glucan levels below that seen in normal
sera. Yasuoka and colleagues27 demonstrated elevated levels of
beta-glucan in the bronchoalveolar lavage specimens in mice
experimentally infected with Pneumocystis carinii but not in
the lavage specimens from uninfected mice. They confirmed
these findings in humans with Pneumocystis pneumonia who
demonstrated significantly higher concentrations of beta-glu-
can compared to the lavage specimens obtained from patients
with other lung disorders. In a recent case report of a patient
with Candida lusitaniae joint infection, beta-glucan test-
ing demonstrated high concentrations corresponding to the
positive cultures.28 Studies in rabbits infected with Candida
albicans to create an experimental model for hematogenous
candidal meningoencephalitis found that 100% of the animals
had demonstratable levels of beta-glucan in the cerebrospinal
fluid.29 In animals cleared of the infection, levels of beta-
glucan in the cerebrospinal fluid decreased as a marker for
response to therapy. Nakao and colleagues30 assayed homog-
enized rat organs to determine the beta-glucan concentrations
found in uninfected organs. Glucan was detected in large
amounts in stool, and in lesser amounts in the small intestine
and lung. The remaining organ homogenates tested (spleen,
kidney, vessels, thymus, liver, and heart) all had very little
detectible beta-glucan in the uninfected rat. These early stud-
ies using beta-glucan testing on specimens other than serum
certainly warrant further investigation and validation before
being widely applied for use in humans.
Summary
The advent, approval, and use of the serum (1-3)-β-D-
glucan assay have changed the testing practices for patients at
risk of developing or patients with invasive fungal infections.
The current FDA-approved kit, Fungitell, is intended to be
used for the detection of serum (1-3)-β-D-glucan as an aid in
the diagnosis of invasive mycoses. The current guidelines of the
Infectious Diseases Society of America for the management of
684 LABMEDICINE ■ Volume 42 Number 11 ■ November 2011 labmedicine.com
Table 4_Proposed Recommendations for the Use of the
(1-3)-β-D-Glucan Assay
1.  Testing should be used in conjunction with other methods for the diagnosis
   of invasive fungal infections
2.  Testing should precede antifungal therapy.
3.  Twice-weekly testing should be used for surveillance of infections in at-risk
   patients.
4.  Once-weekly testing should be used to assess the response to treatment.
5.  Positive test results should be confirmed with a second new specimen or
   repeated from the initial specimen prior to acceptance as a true positive.
6.  Further study is needed to determine the usefulness of testing on specimens
   other than serum.
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candidiasis and aspergillosis recommend serum (1-3)-β-D-
glucan testing to assist in the assessment of patients with
suspected deep-seated fungal infections but offer no addi-
tional recommendations for the specific use of the assay.31,32
However, to provide accurate testing results the laboratory
must be proficient in the performance and quality control of
the assay. In addition, physicians must be familiar with the
limitations of the assay and use it appropriately in patient
management. No proficiency test specimens are available
commercially, so laboratories performing this assay must par-
ticipate in an alternative proficiency testing program. While
no formal guidelines for the use of (1-3)-β-D-glucan testing
have been proposed, using current evidence, proposed testing
recommendations are summarized in Table 4. The current
evidence suggests that a negative test cannot fully rule out the
diagnosis of an invasive fungal infection, while a positive test
alone lacks sufficient sensitivity and specificity for a definitive
diagnosis. The availability of a rapid, easy-to-perform assay
to serve as a surrogate marker for fungal infections is being
embraced for testing at-risk populations, but further trials are
needed to determine the optimal extent and frequency of the
testing that will ultimately be recommended. Further evaluation
is also warranted to determine the usefulness of beta-glucan
testing on specimens other than serum as markers for invasive
fungal disease or response to therapy. LM
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