POINT-COUNTERPOINT

␤-D-Glucan Testing Is Important for Diagnosis of Invasive Fungal

Infections
Elitza S. Theel,a Christopher D. Doernb
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA,a University of Texas Southwestern Medical
Center, Dallas, Texas, USAb

Invasive fungal infections are a significant cause of morbidity and mortality in patients who receive immunosuppressive ther-
apy, such as solid organ and hematopoietic stem cell transplant (HSCT) recipients. Many of the fungi associated with these infec-
tions are angioinvasive and are best diagnosed by visualizing the organism in or culturing the organism from deep tissue. How-
ever, obtaining such tissue often requires an invasive procedure. Many HSCT recipients are thrombocytopenic, making such
procedure too risky because of potential bleeding complications. Additionally, positive blood cultures are rare for patients with
angioinvasive fungal infections, making this diagnostic strategy of little value. Undiagnosed fungal infections in these patient
populations are a significant cause of mortality. Prophylactic use of antifungal agents, such as the echinocandins, during periods
of neutropenia or graft-versus-host disease may prevent some fungal infections but increase the risk for others. Detection of fun-
gal antigens in body fluids, including cryptococcus capsular polysaccharide, histoplasma antigen, galactomannan, and ␤-D-glu-
can, is viewed as being clinically useful for at least the presumptive diagnosis of invasive fungal infections. ␤-D-Glucan is an at-
tractive antigen in that it is found in a broad range of fungal agents, including the commonly encountered agents Candida spp.,
Aspergillus spp., and Pneumocystis jirovecii. Cross-reactions with certain hemodialysis filters, beta-lactam antimicrobials, and
immunoglobulins, which raise concerns about false-positive tests, have also been described. As a result, the use of this testing
must be closely monitored. In this point-counterpoint, we have asked Elitza Theel, who directs the Infectious Disease Serology
Laboratory at the Mayo Clinic, to address why she believes that this test has value in the diagnosis of invasive fungal infections.
We have asked Christopher Doern, Director of Clinical Microbiology at Children’s Medical Center of Dallas, why he questions
the clinical value of ␤-D-glucan testing.

POINT procedures may, however, be counterindicated due to profound

neutropenia, hypoxia, or the overall critical state of the patient.
A n abundant cell wall polysaccharide, (1-3)-␤-D-glucan
(BDG) is found in most fungi, with the notable exception of
the cryptococci, the zygomycetes, and Blastomyces dermatitidis,
Furthermore, even if specimens are acquired, supportive labora-
tory evidence of infection is not guaranteed. Fungal culture from
which either lack the glucan entirely or produce it at minimal lower respiratory tract sources is notoriously insensitive, with a
levels. At least four BDG detection assays have been developed: positivity rate of only 45 to 60% for cases of invasive aspergillosis
Fungitell (Associates of Cape Code, Inc., East Falmouth, MA, (1). Depending on the inoculum and fungal growth characteris-
USA), Wako (Wako Pure Chemical Industries, Ltd., Tokyo, Ja- tics, culture requires at least 2 to 3 days of incubation and, for
pan), Fungitec-G (Seikagaku, Kogyo, Tokyo, Japan), and Maruha some species, days to weeks longer, which may further delay ini-
(Maruha-Nichiro, Foods Inc., Tokyo, Japan), of which only the tiation of antifungal therapy. Positive cultures from nonsterile
Fungitell assay is FDA approved for use on serum in the United sources, including BAL fluid specimens, also require cautious in-
States. This assay is a chromogenic, quantitative enzyme immu- terpretation in order to differentiate between fungal colonization
noassay (EIA) designed to detect BDG (in ng/ml) by using puri- and isolation of the true invasive agent. Finally, fungal blood cul-
fied, lysed horseshoe crab (Limulus polyphemus) amebocytes. tures, while noninvasive and highly specific, require prolonged
These cells contain components of the Limulus clotting cascade, incubation and can likewise be insensitive, with only 50% of Can-
including factors C and G, which initiate coagulation in the pres- dida spp. and ⬍10% of Aspergillus spp. being detected (1, 2). Sole
ence of bacterial liposaccharide and BDG, respectively. By elimi- reliance on culture can therefore lead to delayed diagnosis, and so
nating factor C from the lysate, the manufacturers limit activation a need exists for additional testing methods.
of the cascade to BDG alone. A key advantage of evaluation for BDG is the required speci-
For the purposes of this discussion, the following are argu- men source, serum. A readily available and easily accessible spec-
ments in favor of BDG testing and BDG’s role as a surrogate
marker for invasive fungal infections (IFIs). The caveat remains,
however, that the interpreting clinician must be cognizant of the Published ahead of print 12 July 2013
associated assay limitations. Address correspondence to Elitza S. Theel, [email protected], or Christopher
Readily available specimen source—serum. Currently, the D. Doern, [email protected].
gold standard methods for laboratory-based diagnosis of IFIs in Copyright © 2013, American Society for Microbiology. All Rights Reserved.
patients presenting with pulmonary insufficiency require testing doi:10.1128/JCM.01737-13
of invasively collected specimens, including culture of bronchoal- The views expressed in this feature do not necessarily represent the views of the journal
veolar lavage (BAL) fluid or submission of biopsy material for or of ASM.
histopathologic examination and fungal culture. The invasive

3478 jcm.asm.org Journal of Clinical Microbiology p. 3478 –3483 November 2013 Volume 51 Number 11
 Point-Counterpoint

imen, regardless of patient status, serum allows for serial BDG of limited value and need to be considered in light of available clinical
analysis, which can significantly enhance the assays’ clinical per- and laboratory data. Secondly and perhaps more importantly, these
formance (discussed below). As with testing for other fungal an- studies indicate that among patients with prolonged neutropenia
tigens, detection of BDG in alternative, invasively collected spec- who present with symptoms consistent with an IFI, repeatedly posi-
imens (i.e., BAL fluid and cerebrospinal fluid [CSF]) has the tive BDG results may be used as supportive evidence for the presence
potential to further enhance the sensitivity of standard laboratory of an IFI. This conclusion is further supported by the guidelines of the
practices for IFI diagnosis. Detailed studies evaluating this testing 3rd European Conference on Infections in Leukemia, which catego-
option, however, are still needed. rized BDG testing as “B II,” indicating that there is “moderate evi-
Good performance using serial testing of high-risk patients. dence to support recommendation for use” in patients with leukemia
An initial, overarching review of the BDG literature may lead (5, 16). The EORTC/MSG guidelines, while not used for clinical di-
many readers to completely discount the utility of this biomarker agnosis, have also recently included a positive BDG result as meeting
due to inconsistent performance characteristics. Sensitivity and their criteria for mycological evidence of infection. Currently, how-
specificity values, regardless of the invasive organism, can range ever, neither the ECIL 3 nor the EORTC/MSG provides BDG timing
from 38% to 100% and 45% to 99%, respectively, with similar or interval testing guidelines, and studies to better define serial BDG
ranges observed for the positive predictive value (PPV; 30% to analysis are needed.
89%) and negative predictive value (NPV; 73% to 97%) (3–11). BDG positivity prior to alternative testing methods. A num-
These widely dispersed statistics can be attributed largely to het- ber of groups have now reported that among critically ill patients
erogeneity both within and between evaluations, which differ with with proven or probable IFIs, many will develop detectable BDG
respect to which BDG assay was evaluated, what positive cutoff antigenemia prior to the onset of clinical symptoms or radiologic
criteria was used, the patient and control population tested, and signs or the return of positive culture results. The percentages of
the number of BDG tests performed per individual. The vast ma- patients in whom this occurs vary between studies (64 to 87%), as
jority of these studies appropriately applied the European Orga- do the numbers of days between BDG and culture (blood, biopsy,
nization for Research and Treatment of Cancer/Mycoses Study or BAL fluid) positivity (1 to 10 days) (6–9, 17). While these stud-
Group (EORTC/MSG) criteria to stratify patients with proven/ ies are limited by the number of enrolled patients, the findings
probable/possible or no IFI as the comparator groups against argue that a single positive BDG result should not be haphazardly
which BDG results were evaluated. However, while these guide- discounted. Instead, among patients with a high pretest probabil-
lines remain the sole standard, they can lead to overcalling cases of ity of developing an IFI (which was hopefully the impetus for
possible fungal pneumonia and completely miss autopsy-proven initial BDG evaluation), a single positive BDG test warrants close
IFIs (12, 13). Despite these limitations, careful dissection of the patient monitoring and further clinical and, if possible, laborato-
data reveals certain scenarios where testing for BDG antigenemia ry-based evaluation.
is relevant and may lead to improved patient outcome. Trending of BDG levels may be used to monitor responses to
Two populations that have been shown to consistently benefit therapy. In addition to monitoring qualitative BDG results during
from BDG testing, particularly during episodes of neutropenia, interval testing, tracking quantitative values following initiation of
are patients with hematologic malignancies and those who have antifungal therapy may be used as a prognostic marker for patient
undergone allogeneic hematopoietic stem cell transplants (6, 7, 9, response. Consistently decreasing BDG levels during treatment
14). Prospective, serial BDG antigenemia testing (at least bi- have been shown by multiple groups to result in a favorable ther-
weekly) in these patients, starting at the onset of neutropenia apeutic responses among patients with proven or probable IFIs (6,
(⬍500 cells/mm3), has led to significantly higher specificities 7, 17, 18). Perhaps among the most alluring of these studies is that
(76% to 99%) and NPVs (87% to 96%) for the presence of proven of Jaijakul and colleagues, who plotted serial BDG levels collected
or probable IFI than single-time-point testing. Unfortunately, de- over time from 203 patients with proven invasive candidemia dur-
spite interval testing, the sensitivities and PPVs of the BDG assays ing anidulafungin treatment. Using this charting method, the au-
remain unacceptably low. An intriguing meta-analysis by Lamoth thors correlated a negative slope in BDG levels from patients with
and colleagues, which included six cohort studies, recently re- a favorable treatment outcome (PPV of 90%) and a positive slope
ported a diagnostic odds ratio of 111.8 versus 16.3 for the presence following treatment failure (NPV of 90%) (18). Interestingly,
of IFIs in neutropenic hemato-oncological patients following two among those who responded to treatment and showed a negative
consecutively positive BDG assays compared to a single positive BDG slope, only 16% had a negative BDG result upon endpoint
BDG assay. This meta-analysis also reported a pooled sensitivity of testing. This is not entirely surprising, as the precise kinetics of
49.6%, alongside a PPV and NPV of 83.5% and 94.6%, respec- release and the route of BDG elimination remain unclear. What
tively (4). needs to be underscored, however, is that while monitoring trend-
A number of conclusions can be drawn from the aforemen- ing of BDG values over time can be a useful prognostic marker for
tioned data. First, due to the consistently low sensitivity reported response to treatment, the presence or absence of BDG should not
among studies, and despite the strong NPV, a negative BDG result be used to guide cessation of therapy or as a “test of cure.”
should not be used to exclude the possibility of invasive fungal BDG detection as an aid for diagnosis of Pneumocystis ji-
disease. The lower sensitivity of the BDG assay, however, is not rovecii pneumonia. Immunosuppressed populations are at risk
unique among fungal biomarkers. A meta-analysis of 27 studies for infection with Pneumocystis jirovecii, in addition to invasive
evaluating the performance of the galactomannan (GM) assay disease with Aspergillus or Candida. Pneumocystis pneumonia
among patients with hematologic malignancies identified a pooled (PCP) classically presents with dry cough, dyspnea, and fever in
sensitivity of 61% for patients with proven or probable invasive asper- the setting of diffuse ground glass opacities on chest X ray, and
gillosis (15). Therefore, as with other serologic tests for fungal anti- while characteristic, these symptoms remain broad and can be
gens associated with IFIs, single negative results from BDG assays are induced by a diverse range of microbial pathogens. As with the

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␤-D-Glucan Testing Is Important for Fungal Diagnosis

diagnostic challenges of IFIs, the preferred specimens for detec- 4. Lamoth F, Cruciani M, Mengoli C, Castagnola E, Lortholary O, Rich-
tion of P. jirovecii are BAL fluid or biopsy material obtained by ardson M, Marchetti O. 2012. ␤-Glucan antigenemia assay for the diag-
video-assisted thoracoscopic surgery (VATS), which may be un- nosis of invasive fungal infections in patients with hematological malig-
nancies: a systematic review and meta-analysis of cohort studies from the
attainable at presentation due to concerns for patient safety. Fur- Third European Conference on Infections in Leukemia (ECIL-3). Clin.
thermore, diagnostic procedures, including microscopy of stained Infect. Dis. 54:633– 643.
specimens, can be insensitive, while molecular methods may de- 5. Marchetti O, Lamoth F, Mikulska M, Viscoli C, Verweij P, Bretagne S.
tect low-level, noncontributory colonization. As with other fungal 2012. ECIL recommendations for the use of biological markers for the
pathogens, BDG is a major component of the P. jirovecii surface diagnosis of invasive fungal diseases in leukemic patients and hematopoi-
structure and has been considered a potential marker for PCP. etic SCT recipients. Bone Marrow Transplant. 47:846 – 854.
6. Senn L, Robinson JO, Schmidt S, Knaup M, Asahi N, Satomura S, Mat-
Recently, a meta-analysis evaluating 11 retrospective studies of suura S, Duvoisin B, Bille J, Calandra T, Marchetti O. 2008. 1,3-␤-D-
patients with laboratory-confirmed PCP and at-risk patient con- Glucan antigenemia for early diagnosis of invasive fungal infections in neu-
trols found a pooled sensitivity and specificity of 94.8% and tropenic patients with acute leukemia. Clin. Infect. Dis. 46:878 – 885.
86.3%, respectively, for detection of BDG in cases of proven PCP 7. Ellis M, Al-Ramadi B, Finkelman M, Hedstrom U, Kristensen J, Ali-
(19). Additionally, this group reported a diagnostic odds ratio of Zadeh H, Klingspor L. 2008. Assessment of the clinical utility of serial
113.7 for the presence of PCP in the setting of a positive BDG beta-D-glucan concentrations in patients with persistent neutropenic fe-
ver. J. Med. Microbiol. 57:287–295.
result. In light of the lower specificity and despite multiple reports 8. Del Bono V, Delfino E, Furfaro E, Mikulska M, Nicco E, Bruzzi P,
of significantly elevated, quantitative BDG values among patients Mularoni A, Bassetti M, Viscoli C. 2011. Clinical performance of the
with PCP compared to values for patients with other IFIs, BDG (1,3)-beta-D-glucan assay in early diagnosis of nosocomial Candida
remains a pan-fungal biomarker and positive results require clin- bloodstream infections. Clin. Vaccine Immunol. 18:2113–2117.
ical correlation for a PCP diagnosis to be made. However, the high 9. Odabasi Z, Mattiuzzi G, Estey E, Kantarjian H, Saeki F, Ridge RJ, Ketchum
sensitivity coupled with a strong NPV (⬎95%) identified in indi- PA, Finkelman MA, Rex JH, Ostrosky-Zeichner L. 2004. ␤-D-Glucan as a
diagnostic adjunct for invasive fungal infections: validation, cutoff develop-
vidual studies (20) collectively indicate that a negative BDG result ment, and performance in patients with acute myelogenous leukemia and
may be used to downgrade P. jirovecii as a likely cause of infection. myelodysplastic syndrome. Clin. Infect. Dis. 39:199 –205.
Serial BDG testing may be cost-effective. A natural concern 10. Pickering JW, Sant HW, Bowles CA, Roberts WL, Woods GL. 2005.
that arises when any assay is recommended to be performed at Evaluation of a (1¡3)-beta-D-glucan assay for diagnosis of invasive fun-
multiple intervals is cost. As with most serologic assays, testing of gal infections. J. Clin. Microbiol. 43:5957–5962.
multiple samples (i.e., acute- and convalescent-phase sera) is pre- 11. Mohr JF, Sims C, Paetznick V, Rodriguez J, Finkelman MA, Rex JH,
Ostrosky-Zeichner L. 2011. Prospective survey of (1¡3)-beta-D-glucan
ferred, and detection of BDG should not be considered any differ- and its relationship to invasive candidiasis in the surgical intensive care
ently. Cost per BDG assay can vary (depending on contracts, the unit setting. J. Clin. Microbiol. 49:58 – 61.
performing laboratory, etc.) but typically ranges between $100 12. Subira M, Martino R, Rovira M, Vazquez L, Serrano D, De La Camara R.
and $200. While not inexpensive, considering the economic bur- 2003. Clinical applicability of the new EORTC/MSG classification for invasive
den of prolonged hospitalization in intensive care units, which can pulmonary aspergillosis in patients with hematological malignancies and au-
topsy-confirmed invasive aspergillosis. Ann. Hematol. 82:80 – 82.
quickly mount into the tens of thousands of dollars, serial BDG
13. Obayashi T, Negishi K, Suzuki T, Funata N. 2008. Reappraisal of the
testing has the potential to significantly decrease patient cost. serum (1¡3)-beta-D-glucan assay for the diagnosis of invasive fungal in-
When used in the appropriate setting, repeatedly positive BDG fections—a study based on autopsy cases from 6 years. Clin. Infect. Dis.
results and/or increasing BDG levels may prompt sooner initia- 46:1864 –1870.
tion of broad antifungal therapy and result in quicker resolution 14. Kawazu M, Kanda Y, Nannya Y, Aoki K, Kurokawa M, Chiba S,
or even prevention of severe disease. Detailed studies evaluating Motokura T, Hirai H, Ogawa S. 2004. Prospective comparison of the
diagnostic potential of real-time PCR, double-sandwich enzyme-linked
the potential cost savings for BDG testing are needed, however, for
immunosorbent assay for galactomannan, and a (1¡3)-beta-D-glucan
this to be conclusively established. test in weekly screening for invasive aspergillosis in patients with hemato-
Conclusions. Detection of BDG antigenemia can be a useful logical disorders. J. Clin. Microbiol. 42:2733–2741.
diagnostic tool if used in the proper clinical setting (i.e., immuno- 15. Pfeiffer CD, Fine JP, Safdar N. 2006. Diagnosis of invasive aspergillosis
suppressed, neutropenic patients) by a provider knowledgeable of using a galactomannan assay: a meta-analysis. Clin. Infect. Dis. 42:1417–
both the advantages and limitations of the assay as applied to each 1427.
16. Metan G, Koc AN, Atalay A, Kaynar LG, Ozturk A, Alp E, Eser B. 2012.
individual patient. BDG detection will not replace current labora-
What should be the optimal cut-off of serum 1,3-beta-D-glucan for the
tory methods for IFI diagnosis, and questions remain regarding detection of invasive pulmonary aspergillosis in patients with haemato-
appropriate clinical use (i.e., timing of specimen collection, dura- logical malignancies? Scand. J. Infect. Dis. 44:330 –336.
tion of testing, meaning of quantitative values, etc.). However, the 17. Pazos C, Ponton J, Del Palacio A. 2005. Contribution of (1-⬎3)-beta-D-
ease of specimen collection and the potential information that can glucan chromogenic assay to diagnosis and therapeutic monitoring of inva-
be garnered from serial BDG evaluations argue for consideration sive aspergillosis in neutropenic adult patients: a comparison with serial
screening for circulating galactomannan. J. Clin. Microbiol. 43:299 –305.
of this assay as a diagnostic screen in many diagnostic protocols.
18. Jaijakul S, Vazquez JA, Swanson RN, Ostrosky-Zeichner L. 2012. (1,3)-
Elitza S. Theel ␤-D-Glucan as a prognostic marker of treatment response in invasive can-
didiasis. Clin. Infect. Dis. 55:521–526.
REFERENCES 19. Karageorgopoulos DE, Qu JM, Korbila IP, Zhu YG, Vasileiou VA,
1. Singh N, Paterson DL. 2005. Aspergillus infections in transplant recipi- Falagas ME. 2013. Accuracy of beta-D-glucan for the diagnosis of Pneu-
ents. Clin. Microbiol. Rev. 18:44 – 69. mocystis jirovecii pneumonia: a meta-analysis. Clin. Microbiol. Infect.
2. Ellepola AN, Morrison CJ. 2005. Laboratory diagnosis of invasive candi- 19:39 – 49.
diasis. J. Microbiol. 43:65– 84. 20. Held J, Koch MS, Reischl U, Danner T, Serr A. 2011. Serum (1 ¡
3. Karageorgopoulos DE, Vouloumanou EK, Ntziora F, Michalopoulos A, 3)-beta-D-glucan measurement as an early indicator of Pneumocystis ji-
Rafailidis PI, Falagas ME. 2011. ␤-D-Glucan assay for the diagnosis of rovecii pneumonia and evaluation of its prognostic value. Clin. Microbiol.
invasive fungal infections: a meta-analysis. Clin. Infect. Dis. 52:750 –770. Infect. 17:595– 602.

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 Point-Counterpoint

COUNTERPOINT patients with hematologic malignancy and found the sensitivity

and specificity of the assay to be 100 and 90%, respectively (5).
When the error rate of a test exceeds the prevalence of the Regardless of patient population, a significant limitation of the
disease it is designed to detect, then you don’t have much negative predictive value of BDG testing is its inability to diagnose
of a test. Cryptococcus and zygomycete disease. Even in those patients for
—Gary V. Doern (personal communication) whom sensitivity could be maximized, a negative result does not
exclude the possibility of disease by these important pathogens.

T he concept of the (1,3)-␤-D-glucan (BDG) test is highly desir-

able; it provides a noninvasive test method which is designed
to diagnose invasive fungal infections (IFIs). As more patients
The issue of poor sensitivity is further complicated by the un-
certainty surrounding what value truly defines a positive result.
The three primary providers of BDG testing all interpret their
experience prolonged immunocompromised periods, IFIs have assays differently, so it is particularly important that physicians
become increasingly common. BDG as a marker for infection ordering these tests be aware of the specific method being used.
holds great appeal because making the definitive diagnosis of IFI One group reported that the Fungitell BDG assay had greater sen-
often requires tissue biopsy or is made at autopsy. In contrast to sitivity than both the Fungitec and the Wako tests (8). One meta-
galactomannan testing, which can be used to diagnose only inva- analysis supported these findings (2). When the tests were strati-
sive Aspergillus (IA) infection, BDG testing is promising because it fied by manufacturer, the Wako test was found to have the lowest
is capable of detecting infections caused by many fungi, excluding sensitivity but did have higher specificity than its competitors.
Cryptococcus and the zygomycetes (1). Differences in assay performance are not surprising considering
This counterpoint will review the limitations of the BDG assay that they differ in ␤-glucan standards used, specimen pretreat-
and argue that while, in principal, the test is promising, in practice, ment methods, and kit lysates (5, 9). In addition, the literature
its poor performance renders it of limited use in establishing a evaluates a wide range of performance cutoff values (3 pg/ml to
diagnosis of invasive fungal infections. What follows is an objec- ⬎500 pg/ml) outside those recommended by the manufacturer.
tive assessment of performance data as well as a discussion of the Not surprisingly, when the threshold for positivity is lowered,
utility of BDG testing in certain clinical situations, such as moni- specificity decreases (10). De Vlieger and colleagues evaluated var-
toring response to treatment. ious Fungitell positivity cutoffs in an autopsy-based study evalu-
Sensitivity and specificity of ␤-D-glucan testing. The value of ating the BDG assay performance for IA (10). They found that in
any laboratory test can be judged by whether the result(s) it pro- ICU patients with confirmed IA, a cutoff value of 80 pg/ml yielded
vides can be trusted and acted upon. The utility of a given test a sensitivity of 85.7% but a specificity of only 36.4%. In a subgroup
result is primarily dependent on the test’s sensitivity and specific- analysis that divided patients into hematology and nonhematol-
ity, but also on the prevalence of disease and the resulting positive ogy patients, a cutoff of 140 pg/ml improved the specificity to
and negative predictive values. Ideally, a test will have both high 77.8% but at the expense of sensitivity, which then dropped to
sensitivity and high specificity. 72.7% in hematology patients. Contrary to the findings of the
The literature is now replete with studies evaluating the clinical meta-analysis discussed above, BDG testing functioned better for
performance of the BDG assay in a wide variety of patient popu- nonhematology patients, with a cutoff of 140 pg/ml and a sensi-
lations. Although significant heterogeneity exists in these studies, tivity and specificity of 100 and 69.6%, respectively (10). In their
one common finding is that the sensitivity of this assay is between final analysis, they state that although patients with IA had higher
50 and 80% and its specificity ranges between 50 and 90% (1–5). BDG levels than those who did not, performance characteristics
One meta-analysis concluded that the average sensitivity and did not justify the test’s use as a diagnostic tool for IA. Due to the
specificity were approximately 76% and 85%, respectively (2). sliding scale of interpretive criteria and the heterogeneity in the
Considering these performance characteristics, the error rate of literature, health care providers are left with the difficult task of
BDG testing far exceeds the prevalence of invasive fungal infec- deciding what positive cutoff to use and how to know when a
tions in most settings in which BDG testing is applied. As a result, negative result really indicates the absence of disease. If individual
BDG testing is often of limited diagnostic value. institutions are inclined to pursue BDG testing in spite of its rela-
Limitations of BDG assay sensitivity. BDG testing is com- tively low sensitivity, they would be well served in conducting
monly utilized in patients that have underlying conditions, such as investigations aimed at defining its true utility in their own patient
organ transplantation or malignancy, and therefore have highly populations.
compromised immune systems. It is in these patients that a test for Limitations of BDG assay specificity. The greatest limitation
IFI with a high sensitivity and, therefore, good negative predictive of BDG testing is its poor specificity. The list of factors which can
value would be of great value. Unfortunately, a literature review generate false-positive results is extensive and includes albumin,
reveals an average sensitivity in the mid-70s and a correspondingly intravenous immune globulin, gauze packing, intravenous
poor NPV (2, 6). Given this poor sensitivity, negative test results amoxicillin-clavulanic acid, and use of cellulose depth filters (11,
do not allow caregivers to confidently exclude invasive fungal dis- 12). It has also been suggested that bacteremia due to Gram-pos-
ease. itive organisms and Alcaligenes faecalis can result in false-positive
Some subanalyses stratified by underlying condition suggest BDG tests. In addition, in vitro studies have demonstrated that
that BDG testing may function better for patients with hemato- colistin, ertapenem, cefazolin, trimethoprim-sulfamethoxazole,
logical malignancy than for solid organ transplant patients or in- cefotaxime, cefepime, and ampicillin-sulbactam can all yield pos-
tensive care unit (ICU) patients (2). However, the data in this itive BDG test results (13). Marty and colleagues examined these
regard are mixed, as Hachem et al. have shown poor performance antimicrobial agents at reconstituted vial concentrations and
in patients with hematologic malignancy (7). To illustrate the het- found BDG reactivity. However, when these agents were diluted to
erogeneity in the literature, Odabasi and colleagues also evaluated what would be considered maximum plasma concentrations, they

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␤-D-Glucan Testing Is Important for Fungal Diagnosis

found no reactivity, suggesting that these compounds might not addresses the use of the BDG assay in clinical practice, it is clear
yield false-positive results in patients receiving these agents ther- that laboratories cannot assume that their physicians are aware of
apeutically (13). There may still be cause for concern though, as at their own patients’ risk factors for a falsely positive BDG result. A
least one study found elevated BDG values in 37 of 117 serum number of retrospective studies looking at BDG test use show that
samples from patients being treated with ampicillin-sulbactam it is quite common for this test to be ordered for those with risk
and no other explanation for the false-positive result (11). factors for false positivity (3).
The limitations of BDG cross-reactivity have been well docu- The value of multiple positive tests. Some studies and reviews
mented in the literature. What are less well described are the ad- suggest that the BDG assay has a high negative predictive value
verse events that almost certainly result from these false positives. when multiple negative tests are considered. It is difficult to deter-
Of primary concern is the misdiagnosis of IFIs, leading to unnec- mine a consensus negative predictive value because the literature
essary antifungal treatment and distraction from diagnosing the is very heterogeneous and prevalence rates vary widely and are
actual cause of a patient’s disease. The poor specificity is particu- greatly influenced by study design. However, a meta-analysis con-
larly problematic in immunocompromised patients, in whom it is cluded that the sensitivity of two consecutive positive tests was
most commonly used. With the high morbidity and mortality 65%, with a 95% confidence interval of 52 to 78% (2). Although
associated with IFIs, it is very difficult to justify withholding anti- the sensitivity of this approach is poor, the authors did find that
fungal treatment for a test result that may suggest invasive disease. two consecutive positives yielded a specificity of 93%. Senn et al.
This leads to excessive use of antifungal agents and all of the neg- published an evaluation of serial BDG testing and various cutoff
ative consequences associated with inappropriate usage. Indeed, values using the Wako test (17). In this study, when the lowest
resistance among fungal pathogens is becoming more common in cutoff values were applied, two consecutive positives generated a
the face of increased exposure (14). Some studies have now shown sensitivity of 97% but a specificity of 51%. Conversely, when the
that the MICs for Aspergillus isolates increase as the isolates are highest cutoff values were used, two consecutive positives gener-
exposed to increasing concentrations of triazoles (15). What is ated a specificity of 99% but with only a 40% sensitivity. Neither of
particularly concerning is that exposure to one triazole correlated these scenarios is acceptable for clinical care. When a middle cut-
positively with increased MICs of different triazoles (15). off value was applied to maximize overall diagnostic efficiency, the
Clearly, the less-than-optimal specificity of BDG assays creates sensitivity was 63% and the specificity was 96%. The authors did
a dilemma for health care providers who have to make treatment go on to calculate the positive and negative predictive values, but
decisions for patients who are critically ill. The negative outcomes the prevalence in this study was high, which makes it difficult to
associated with BDG testing have yet to be quantified. However, draw strong conclusions from these calculations. It is interesting,
one ICU-based study prospectively evaluated the use of BDG test- though, that even in the face of a 31% prevalence, the positive
ing for deciding when to preemptively treat patients for IFIs. In predictive value of two consecutive tests was still only 79%. If we
that study, patients in the intervention arm were preemptively extract their sensitivity and specificity calculations and insert the
treated with anidulafungin if BDG testing was positive. Of rele- prevalence obtained by other prospective studies of 10%, the pos-
vance to the specificity concerns, the positive predictive value of a itive predictive value drops to 62% (4, 17).
positive BDG test in that study was only 30%. In other words, 70% Conclusions. Although the BDG assay fills a void in diagnostic
of the time, BDG testing yielded a positive result and it was incor- testing for invasive fungal infection in the immunocompromised,
rect. The study was different than other retrospective studies, it does so with poor sensitivity and specificity. The sensitivity is
which may inflate prevalence due to preselection and therefore not sufficient to confidently rule out fungal infection, and the
exaggerate the positive predictive value of BDG positivity. One poor specificity makes it very difficult to interpret positive results.
other study of patients with hematological malignancies undergo- The frequently generated false-positive results are particularly
ing treatment had specimens prospectively collected but retro- concerning, as they may be difficult to ignore even in patients with
spectively tested. In this study, the prevalence of IFI was found to known risk factors for false positivity. In worse-case scenarios,
be 8.7%, resulting in a positive predictive value of only 12% (16). patients awaiting transplants may be committed to long periods of
One could make the case that if you were aware of all the dif- antifungal treatment until a presumed fungal infection has been
ferent conditions which may lead to BDG false positivity, you completely treated, thus delaying a potentially life-saving trans-
could simply avoid ordering the test for those patients. The prob- plant. There is a growing body of evidence that BDG performs
lem with this approach is that the full extent of BDG cross-reac- with high sensitivity for patients with Pneumocystis jirovecii pneu-
tivity has not been determined. Indeed, when Koo et al. evaluated monia (PJP); however, all of the same limitations in specificity still
BDG performance in stem cell transplant patients, they found that apply. That said, the test may serve as a suitable screen for PJP
excluding those patients with risk factors for BDG false positivity infection, but providers should be aware that it is only a good
did not significantly improve its specificity (3). Another study by rule-out test and that positive results should be confirmed with a
Bellanger et al. (18) found that 4 of 11 hematology control patients Pneumocystis-specific assay.
had false-positive BDG tests despite the preselection of patients In conclusion, despite the desperate need for improved diag-
with no known risk factor for BDG cross-reaction. Lastly, in the nostics of invasive fungal infections, detection of BDG does not
Racil et al. study mentioned above, a thorough analysis of the appear to be a test with sufficient sensitivity or specificity to be
patients was conducted in an attempt to explain a false-positivity recommended for routine use.
rate of ⬎50% (16). They were unable to identify known risk fac- Christopher D. Doern
tors for false positivity. The erroneous results appeared to happen
at random and without explanation. This may suggest that there REFERENCES
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SUMMARY
Points of agreement
• This test is likely of value only for select patient populations (HSCT patients) or in the diagnosis of select infections, such as with
P. jirovecii pneumonia.
• Multiple positive results are best at predicting the presence of invasive fungal infections.
• Because of many potential sources of false positives, a single positive test is of limited value.
• The test has poor sensitivity, so it has no value as a screening test; a negative test cannot exclude the diagnosis of invasive fungal
infection.
• Monitoring ␤-D-glucan quantitative levels in patients being treated for invasive Candida infections has value.
Issues to be resolved
• Further studies are needed to determine the patient populations and clinical scenarios in which this test is most likely to be of
value.
• The frequency with which this test should be performed for diagnosis or therapeutic monitoring is not well understood.
• Detailed studies of the performance of ␤-D-glucan testing with other specimen types, such as cerebrospinal fluid and bronchoal-
veolar lavage (BAL) fluid, are needed. Performance of this test with BAL fluids may be of particular value for detecting P. jirovecii
pneumonia.
• Improvements in the assay to eliminate the high rates of false positives due to commonly encountered materials and therapeutics
would greatly increase its clinical value.
Peter H. Gilligan, Point-Counterpoint Editor, Journal of Clinical Microbiology

November 2013 Volume 51 Number 11 jcm.asm.org 3483