Articles, Blog

Detection of (1-3)-β-D-glucan as a Marker of Invasive Fungal Disease

October 18, 2019


Hi, I’m Bobbi Pritt, Director of the Clinical
Parasitology Lab and Vice Chair of Education in the Department of Laboratory Medicine and
Pathology at Mayo Clinic. Diagnosis of invasive fungal infections, including
invasive aspergillosis can be a challenge, particularly in immunocompromised hosts. In this “Hot Topic,” my colleague, Dr.
Elli Theel, will discuss detection of beta-(1,3)-D-glucan or BDG in serum as a biomarker for the presence
of such invasive fungal infections. As Dr. Theel will discuss, BDG is a cell wall
component of many clinically relevant fungi, including Aspergillus sp, Fusarium sp. and
Pneumocystis among others. I hope you enjoy this month’s Hot Topic,
and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner
in your patient’s health care. Thank you for the introduction and to the
listeners for joining us today to talk about testing for the fungal antigen, 1-3 beta-d-glucan. Before we begin, just a note that I do not
have any relevant financial disclosures to share. Broadly speaking, invasive fungal disease
is defined as a fungal infection of an otherwise sterile site, such as the bloodstream or deep
tissues. While both opportunistic and endemic fungal
pathogens can cause invasive disease in the right host, some of the most common fungal
pathogens causing invasive fungal infections include Candida species., Aspergillus species.,
Pneumocystis and Cryptococcus species., and while the overall incidence of invasive fungal
disease can vary somewhat from region to region, in higher income countries, the incidence
ranges from approximately 14 to 27 cases per 100,000 patients per year. Those at highest risk of IFDs include individuals
having recently received a hematopoeitic stem cell transplant, a solid organ transplant,
those that have significant and prolonged neutropenia and others that are significantly
immunosuppressed due to malignancy, receipt of chemotherapy or on treatment with certain
biologic agents. Importantly, IFDs are associated with significant
morbidity and mortality, particularly in situations where diagnosis and initiation of appropriate
antifungal therapy is delayed. The diagnosis of IFDs is challenging for multiple
reasons, including the fact that symptoms are largely non-specific, and so a combined
diagnostic approach is typically required. This includes imaging studies, and while certain
classic radiologic signs like the halo or crescent sign on chest imaging have been well
described, these are infrequently observed. With regards to laboratory testing, the gold
standard remains histopathologic examination of biopsy material from affected tissues alongside
culture, however invasive specimen collection procedures may be contraindicated particularly
in patients with significant respiratory insufficiency. Routine fungal culture can be helpful, however
results may also be confounding. Sensitivity of culture is generally low and
is largely dependent on the quality of the specimen submitted. Additionally, some fungal pathogens require
prolonged incubation, which can further delay diagnosis. Because of these limitations, significant
effort has gone into developing additional or alternative diagnostic assays for detection
of IFDs, including both molecular testing and identification of fungal biomarkers, including
detection of the pan-fungal biomarker 1-3 beta-d-glucan or BDG, which is the focus of
today’s presentation. BDG is a polysaccharide composed of glucose
monomers linked by beta 1-3 glucosidic bonds and is found in high abundance in cellulose
containing plants, and it also makes up a significant proportion of the cell wall of
many fungi, including clinically important fungi like Aspergillus, Candida, Fusarium,
and others. It is actually easier to remember which organisms
lack BDG or produce it in low amounts, which include the Mucorales agents, such as Mucor
species and Absidia species, and also Cryptococcus species and Blastomyces dermatitidis. There are at least five commercially available
assays for detection of BDG, however only the Fungitell assay from the Associates of
Cape Cod is FDA-approved for detection of BDG in serum, with the intended use as an
aid in the diagnosis of deep-seated IFDs. It is also important to remember that results
from this assay need to be used in conjunction with the other diagnostic procedures previously
mentioned, including imaging studies, culture and histopathologic examination of biopsy
material as available. TThe Fungitell BDG assay is a chromogenic,
quantitative EIA based on the clotting cascade of the Limulus or horseshoe crab. Briefly, horseshoe crab amebocytes are isolated
and lysed to release components of the crab clotting cascade and it is this lysate that
is used for the Fungitell assay. This clotting cascade can be stimulated by
two different molecules, either by the presence of bacterial endotoxin or by BDG, as shown
here. In order to make this assay specific for detection
of BDG, factor C has been eliminated by the manufacturer, which is the component that
activates the clotting cascade in the presence of endotoxin. So with this modification, in the presence
of BDG, Factor G, a serine protease zymogen, is activated, which activates the clotting
enzyme which can then cleave this externally added chromogenic peptide substrate to release
para-nitroaniline or pNA, which is excited at a specific wavelength. Unlike most other standard ELISAs, this assay
is a kinetic ELISA, meaning that each well for each patient sample, which is run in duplicate,
is read and optical density or OD values recorded every 30 seconds over a 40 minute period. As an example, this is what a single patient
run looks like after completion. Each run has BDG standards run in duplicate,
alongside patient sera, also run in duplicate. The OD values are collected at each 30 sec
interval and are plotted over the 40 minute time frame to generate a curve. Interpretation of results involves both analysis
of the curve shape as well as software analysis to establish the mean rate of change in the
OD, which is then used to determine the final BDG concentration value in pg/mL using the
standard curve. Without going into details, positive patient
results typically have ‘hockey stick’ shaped curves with a notable increase in OD
levels over time. This is in comparison to samples with negative
results which typically have straight curves and a negligible change in OD values over
the 40 minute period. Additionally, some samples may be reported
as ‘uninterpretable due to optical artifact’. In these samples, although there is a significant
change in the OD values over time, the associated curves are aberrant as shown in this example. Various factors may lead to such a result,
and repeat testing of a new specimen may be helpful. Finally, the quantitative range of the Fungitell
assay is 31 to 500 pg/mL, with qualitative interpretive cut-offs of 80 pg/mL or greater
considered positive, 59 pg/mL or less considered negative and indeterminate results range in
value from 60 to 79 pg/mL. The performance characteristics of BDG assays
for detection of IFDs vary significantly in the literature, and importantly multiple studies
and meta-analyses have shown that these assays perform best in patients that are at high
risk for the presence of IFDs as indicated here. Findings from 4 different meta-analyses performed
over the years are summarized in the table below and show that in patients at high-risk
of IFD, single time point beta-d-glucan testing is associated with a sensitivity and specificity
generally ranging between 60 and 90%. Interestingly, multiple studies, performed
primarily in patients with hematologic malignancies, have shown that the presence of two consecutively
positive BDG results increase specificity of the assay to almost 99%, suggesting that
these results may be used as a diagnostic marker for the presence of an IFD. Additionally, it is important to remember
that the absence of BDG antigenemia should be interpreted with caution as a single negative
result may not be used to rule out IFD, and this is true for most fungal pathogens with
the notable exception of IFD due to Pneumocystis. Very briefly, as you’ll remember, Pneumocystis
jirovecii is a fungus, which notably does not respond to antifungals, and is associated
with significant mortality in immunosuppressed patients. Similar to other fungal infections, diagnosis
of Pneumocystis can be challenging, which is further hampered by its inability to be
grown in culture. Notably however, BDG is a major component
of the Pneumocystis cell wall, and an 11 study meta-analysis performed in 2013 showed that
the overall sensitivity and specificity of the Fungitell assay in patients with Pneumocystis
pneumonia was 95% and 86% respectively. Importantly, the authors also found that the
negative likelihood ratio associated with a negative result was 0.06, collectively indicating
that a negative BDG result can reasonably be used to exclude the diagnosis of Pneumocystis
pneumonia. To conclude, there are a few additional test
utilization pearls to remain cognizant of when considering beta-d-glucan testing. First, beta-d-glucan may be detected in patients
prior to symptom onset and in some cases as early as one week in advance of symptoms. Therefore, a single positive BDG result in
at risk patients does warrant close monitoring and/or further targeted evaluation for an
invasive fungal process. Additionally, although some studies support
the trending of BDG levels as a means to monitor response to therapy, other studies have shown
contradictory data. So, while decreasing titers may be associated
with clinical improvement in cases of invasive candidiasis for example, increasing beta-d-glucan
levels may not necessarily mean worsening disease and results should be interpreted
with caution by clinicians. Finally, it is important to remember that
there are certain treatments and conditions that may lead to detection of beta-d-glucan
in patients without IFD. These include recent infusion of IVIG or albumin,
which are frequently filtered through cellulose containing filters. Also, gauze packing during surgery and hemodialysis
using cellulose containing membranes may lead to leaching of beta-d-glucan into the bloodstream. Certain antibiotics have been implicated with
leading to elevated beta-d-glucan levels as a result of how the antibiotics are prepared,
and finally, elevated beta-d-glucan levels have been documented in bacteremic patients,
and in those with severe mucositis and mucosal colonization with Candida. Therefore, as a result of all of these caveats,
it is of critical importance that beta-d-glucan testing be restricted only to patients at
high risk of IFDs and that results be interpreted alongside other clinical and laboratory findings.

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