This study was supported by an investigator-initiated grant from Pfizer, Inc. There are no other relevant declarations relating to this study and employment, consultancy, patents, or products to disclose. The funding source did not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
Conceived and designed the experiments: KEH JRP BDA. Analyzed the data: KEH JRP BDA. Wrote the paper: KEH JRP BDA. Clinical Data Collection: AKZ CDP EDL. Critical Manuscript review: AKZ CDP EDL AHB. Statistical analysis: AHB.
Invasive candidiasis (IC) is a devastating disease. While prompt antifungal therapy improves outcomes, empiric treatment based on the presence of fever has little clinical impact. Β-D-Glucan (BDG) is a fungal cell wall component detectable in the serum of patients with early invasive fungal infection (IFI). We evaluated the utility of BDG surveillance as a guide for preemptive antifungal therapy in at-risk intensive care unit (ICU) patients.
Patients admitted to the ICU for ≥3 days and expected to require at least 2 additional days of intensive care were enrolled. Subjects were randomized in 3∶1 fashion to receive twice weekly BDG surveillance with preemptive anidulafungin in response to a positive test or empiric antifungal treatment based on physician preference.
Sixty-four subjects were enrolled, with 1 proven and 5 probable cases of IC identified over a 2.5 year period. BDG levels were higher in subjects with proven/probable IC as compared to those without an IFI (117 pg/ml vs. 28 pg/ml; p<0.001). Optimal assay performance required 2 sequential BDG determinations of ≥80 pg/ml to define a positive test (sensitivity 100%, specificity 75%, positive predictive value 30%, negative predictive value 100%). In all, 21 preemptive and 5 empiric subjects received systemic antifungal therapy. Receipt of preemptive antifungal treatment had a significant effect on BDG concentrations (p< 0.001). Preemptive anidulafungin was safe and generally well tolerated with excellent outcome.
BDG monitoring may be useful for identifying ICU patients at highest risk to develop an IFI as well as for monitoring treatment response. Preemptive strategies based on fungal biomarkers warrant further study.
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Invasive candidiasis (IC) is associated with high mortality, prolonged hospitalization and excess costs
Establishing a definitive diagnosis of IC is difficult. The accompanying signs and symptoms are nonspecific and may not be present until the disease is advanced or disseminated. Standard microbiologic methods such as blood culture are time consuming and insensitive for early onset IC. Culture-based treatment is therefore delayed, which in turn fuels poor outcomes
The limitations of empirical and culture-driven antifungal therapy have prompted investigation of alternative ICU strategies. These approaches include targeted prophylaxis
This is a single center, randomized, non-blinded, parallel group pilot study assessing the feasibility and utility of biweekly β-D-glucan testing as a guide for preemptive antifungal therapy in at-risk intensive care unit patients. We also assessed the outcome of biomarker driven preemptive therapy as compared to current practice (i.e., empiric treatment based on physician preference) for the management of IC. Overall, the purpose was to generate preliminary data to inform the design of a future, statistically powered multicenter clinical trial.
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
The study protocol was approved by the Duke University Institutional Review Board.
Between June 2008 and January 2011, adult patients (≥18 years) admitted to the Surgical ICUs for at least 3 days, who were also expected to require ≥2 additional days of intensive care, were screened. Due to slow study enrollment, Medical ICU patients were also considered eligible beginning in August 2009. with intolerance to echinocandins, transaminases ≥10 times normal, visibly icteric serum, lung transplant recipients
Eligible patients providing written informed consent were stratified according to APACHE II score (> or ≤20) and randomized in 3∶1 fashion to receive either active surveillance with preemptive anidulafungin or empiric antifungal therapy. Random allocation sequence was generated using free on-line software (Research Randomizer Version 3.0,
BDG testing was performed at baseline and then twice weekly in the ICU. Results were reported within 24 hours, but baseline measurements were not used to inform initiation of preemptive therapy. Subjects with a subsequent BDG determination of ≥60 pg/ml were preemptively treated with a single course of intravenous anidulafungin (100 mg daily for at least 14 days). The dose and duration of anidulafungin was based on the FDA approved treatment schedule for candidemia. Blood cultures were obtained prior to initiation of preemptive therapy, with additional collections based on clinical indicators of infection. To improve sensitivity as a screening test, a positive BDG cut-off of 60 pg/ml was selected based on the observation that 81% of IC patients will have a positive test at this level
To assess BDG kinetics on therapy, serum specimens were collected every other day while subjects were receiving anidulafungin. Kinetic sampling was batch tested retrospectively and these results were not available for treatment decisions by clinicians.
Subjects in the empiric therapy group had BDG testing on the same schedule, but physicians remained blinded to the results. All antifungal treatment decisions were based on the providers’ clinical judgment, which is standard practice for non-transplant, non-leukemia/lymphoma patients at Duke University.
An oral wash or endotracheal suction for
The criteria used to establish a diagnosis of proven or probable IC are displayed in
A. Proven IC |
1. At least 1 blood culture growing |
2. |
3. Histopathology showing invasive |
|
1. Fever (≥38.5°C) or hypothermia (<35.0°C) with leucocytosis (WBC ≥12 K) and/or hemodynamic instability (MAP <65) otherwise unexplained despite at least 3 days of broad spectrum antibiotics and both of the following: |
a. |
b. No alternative microbiologic diagnosis. |
2. Symptomatic |
3. Endoscopically visualized esophagitis with biopsy exclusion of other causes and evidence of yeast on microscopy or |
The sample size for this “pilot” study was not statistically based. Instead, a feasible sample size of 100 subjects was projected based on the number of eligible patients expected to be admitted to the ICU over a 1 year period. SAS (v9.1 SAS Institute Inc., Cary, NC) and R (v2.14.0 Foundation for Statistical Computing, Vienna, Austria) software packages were used for data analyses. Significance set at the 5% level.
The primary outcome measures of interest were the feasibility of preemptive antifungal therapy, BDG test performance and anidulafungin safety/tolerability in an ICU population. Feasibility was assessed with measures of protocol adherence. BDG test characteristics were evaluated with calculations of sensitivity (SN), specificity (SP), positive/negative predictive values (PPV and NPV) and positive/negative likelihood ratios (LR+ and LR-). Repeated measures logistic regression was applied to assess possible causes of false positive BDG results. Predicative variables included recent major surgery, renal replacement therapy, bacteremia, as well as receipt of commercial blood products and/or β-lactam antibiotics all within a week of BDG collection. Treatment emergent AEs were described with summary statistics.
Secondary outcomes included a comparison of the proportion of subjects developing a proven or probable IFI in the preemptive vs. empiric therapy groups as well as the effect of preemptive anidulafungin on serum BDG concentration over time. Clinical and microbiologic characteristics were compared using the Chi-squared or Fisher’s exact test for discrete variables and Mann-Whitney for continuous variables. The influence of antifungal treatment on BDG concentrations was assessed using non-parametric repeated measures ANOVA. A linear mixed effects model was also fitted, using a random subject intercept and a fixed treatment effect by study day. Bootstrap resampling was used to compare rates of glucan decline over time for preemptively treated versus untreated subjects.
More than 1000 ICU admissions were screened over a 2.5 year period. The trial was stopped in January 2011, short of the targeted 100 subject sample size, due to slow accrual. Subject disposition is illustrated in
Abbreviations: RX = treatment; TX = transplant; LFTs = liver function tests; ULN = upper limit of the normal reference range; IC = invasive candidiasis; IFI = invasive fungal infection; IA = invasive aspergillosis; BDG = β-D-Glucan; medical vs. surgical = ICU location.
Subject demographics are displayed in
Parameter | Enrolled N = 64 [Total (%)] | Preemptive Group N = 47 | Empiric Group N = 17 |
Median age (range in years) | 60 (19–82) | 58 (19–79) | 60 (22–82) |
Male sex | 44 (68.8) | 31 (70.0) | 13 (76.5) |
SICU location | 53 (82.8) | 37 (78.7) | 16 (94.1) |
Median APACHE II (range) | 14 (5–25) | 14 (6–25) | 14 (5–25) |
Central venous catheter | 64 (100) | 47 (100) | 17 (100) |
51 (79.7) | 38 (80.9) | 16 (94.1) | |
Any Surgery | 55 (85.9) | 40 (85.1) | 15 (88.2) |
Intra-abdominal procedure | 12 (18.8) | 9 (19.1) | (17.6) |
Broad spectrum antibiotic | 45 (70.3) | 33 (70.2) | 12 (70.6) |
Beta-lactam | 42 (65.7) | 30 (63.9) | 12 (70.6) |
Diabetes | 29 (45.3) | 23 (48.9) | 6 (35.3) |
Hemodialysis | 5 (7.8) | 4 (8.5) | 1 (5.9) |
Immunosuppressive therapy | 9 (14.0) | 8 (17.0) | 1 (5.9) |
Total parenteral nutrition | 6 (9.4) | 4 (8.5) | 2 (11.8) |
Blood Products | 53 (82.8) | 38 (80.8) | 15 (88.2) |
Acute Pancreatitis | 1 (1.7) | 1 (2.1) | 0 (0) |
ANC < 500 | 0 (0) | 0 (0) | 0 (0) |
Median LOS (range in days) | 17 (5–62) | 19 (5–43) | 15 (6–62) |
≥3 Risk factors for IC | 59 (92.2) | 43 (91.5) | 16 (94.1) |
≥4 Risk factors for IC | 40 (62.5) | 31 (70.0) | 9 (52.9) |
Subject characteristics and baseline invasive candidiasis (IC) risk factors were assessed beginning the week prior to study enrollment, percentages (%) are displayed unless otherwise specified. N = number, SICU = surgical intensive care unit; Colonization was defined as the isolation of
A total of 296 BDG tests were performed. Median time from ICU admission to the first BDG assessment was 8 days (range, 3–20). The median number of tests per subject was 4 (range, 1–13). In all, 34 subjects (55%) developed at least one positive BDG test (≥60 pg/ml) prior to receipt of systemic antifungal therapy and 26 (41%) had at least 2 sequential positive tests at this level. Nineteen (31%) subjects were BDG positive at screening/baseline and 2 of these were only positive at the time of study enrollment. Overall, quantitative BDG values ranged from <31 to 1994 pg/ml.
There was no significant difference in the number of proven/probable cases of IC diagnosed between the preemptive and empiric treatment groups (6.4% [3/47] vs. 17.6% [3/17]; p = 0.47) (
Composite definitions for proven or probable IFI were used as the “gold standard” for assessments of test performance
BDG Test Result Cut-off (pg/ml) | SN % | SP % | PPV % | NPV % | LR(+) | LR(−) |
Single ≥60 | 100 | 50 | 17.6 | 100 | 2 | 0 |
Single ≥80 | 100 | 58.9 | 20.7 | 100 | 2.4 | 0 |
Single ≥100 | 100 | 69.6 | 26.1 | 100 | 3.3 | 0 |
Sequential ≥60 | 100 | 67.9 | 25 | 100 | 3.1 | 0 |
Sequential ≥80 | 100 | 75 | 30 | 100 | 4 | 0 |
Sequential ≥100 | 50 | 78.6 | 20 | 93.6 | 2.3 | 0.6 |
SN = sensitivity; SP = specificity; PPV = positive predictive value; NPV = negative predictive value; LR (+) = positive likelihood ratio; LR (−) = negative likelihood ratio.
The positive predictive value of two sequential β-D-Glucan test results ≥80 pg/ml is plotted relative to increasing invasive candidiasis prevalence. Sensitivity and specificity have been fixed at 100% and 75%, respectively.
Overall, 45% (28/62), 37% (23/62) and 23% (14/62) of evaluable subjects had false positive BDG results when the ≥60, 80, and 80 pg/ml×2 thresholds were applied to case definitions of proven/probable IFI, respectively. There was no clear pattern as to the timing of these false positive BDG results (i.e., early [the first week] vs. late during the ICU admission).
Factors previously associated with false positive BDG results were common in the study population. The majority of subjects received B-lactam antibiotics (67.7%, 42/62) and/or commercial blood products (85.5%, 53/62) at some point during their ICU stay. In addition, 14 patients (22.6%, 14/62) developed a bacterial blood stream infection (BSI) while in the ICU (13 gram positive BSIs and 1 gram negative BSI). A single subject was treated with IVIG. This patient had the highest BDG values overall (range, 568–1994 pg/ml) and had received the infusion 2 days prior to the first positive test. Receipt of hemodialysis, however, was the only clinical factor statistically associated with elevated glucan levels (p<0.0001).
Receipt of preemptive antifungal therapy had a significant effect on median glucan concentrations (p<0.001). However, BDG levels declined in both antifungal treated as well as untreated subjects over time (
The antifungal treatment effect on glucan concentration over time was modeled as a linear trend. Abbreviations: 0 = subjects in the standard care group with at least one positive BDG test, but no systemic antifungal treatment; 2 = subjects in active surveillance group that were treated with preemptive anidulafungin; SE = standard error of the estimated glucan concentration slope.
Twenty-one subjects received preemptive therapy for a median duration of 13 days (range, 2–27 days). In all, 10 (48%) subjects experienced 15 AEs that were possibly related to the study drug. Most AEs (80%, 12/15) were mild in severity, reversible, and did not require cessation of anidulafungin. The most common was elevated LFTs (9 of 21 subjects), followed by hypocalcemia and thrombocytopenia (2 of 21 subjects, each). No serious drug-related adverse events were observed during the study period.
Preemptive protocol adherence was 86.7% (39/45). Four subjects received systemic antifungal therapy in spite of repeatedly negative BDG results (21.1%, 4/19), thus breaking the surveillance strategy. None of the 4 met study criteria for a proven or probable IFI. Antifungal therapy was prescribed for several reasons: 1 subject was diagnosed with polymicrobial sinusitis including yeasts, another developed a cutaneous yeast infection, and 2 were treated empirically for fever in the setting of other culture confirmed infections. Two subjects had preemptive therapy withheld despite non-baseline/screening BDG results of ≥60 pg/ml; but both had been transitioned to comfort care prior to receipt of the test results.
ICU patients have a higher incidence of invasive
Identifying ICU populations that are most likely to benefit from early interventions (i.e. the likelihood of IC exceeds 10%
Assays that detect fungal cell wall components in the blood of patients with early IFI are commercially available and have been used in Japan for many years. Attractive features of the FungitellTM BDG assay as a screening test are its sensitivity for the detection of early infection
Potential causes of false-positive BDG results have been identified and many of these factors are common in the ICU
Overall adherence to the BDG surveillance protocol was good, with > 85% of preemptive subjects managed appropriately. Only 4 subjects assigned to the preemptive group received antifungal therapy despite negative BDG results and repeatedly negative results were used to safely withhold antifungal therapy in the majority of cases (78.9%, 15/19). Given the medical complexity of ICU patients, these observations suggest an acceptance of the BDG-driven strategy and a reliance on the negative predictive value of the test. BDG testing with present assays, however, is relatively expensive and labor intensive. Future studies incorporating biomarkers might consider alternative sampling schemes or the development of more facile assays that are amendable to the workflow of a routine clinical laboratory.
The main limitation of this study is the small number of subjects with proven/probable IC. Despite liberal inclusion criteria, we had a difficult time accruing patients not already receiving systemic antifungal therapy by day 3 of ICU admission. Targeted antifungal prophylaxis is routinely used for Duke solid organ transplant, peripheral blood stem transplant, high-risk leukemia/lymphoma and ventricular assist device patients. Reasons why potential subjects were already receiving systemic antifungal therapy was not documented as a part of the study screening process, however, we suspect that a significant proportion of ineligible ICU patients fell in to one of these categories. This may have biased study enrollment toward lower risk ICU subjects. In addition, we relied heavily on consent authorization obtained from patients’ legal representatives, but only half of all potentially eligible subjects had a designated surrogate available to discuss study participation during regular business hours. In the future, having the capacity to consent and screen both subjects in the evening and on weekends might modestly improve accrual. Perhaps more importantly, consideration should be given to initiating study enrollment/screening at the time of surgical or medical ICU admission, recognizing that some subjects will have a falsely elevated BDG level immediately following surgery. Antifungal therapy would then be withheld as a part of the protocol in selected subjects with a negative BDG test at baseline and during serial measurements.
In conclusion, this pilot study represents a first attempt at preemptive antifungal therapy based on the fungal biomarker BDG in busy tertiary care ICUs. Our results suggest that randomized studies of preemptive antifungal therapy in the ICU are feasible, but that these studies should ideally involve centers that do not routinely utilize fluconazole prophylaxis or employ protocols in which prophylaxis is held for selected patients with negative screening tests. Multicenter trials will ultimately be required to determine the efficacy, optimal algorithm, and cost-effectiveness of preemptive approaches for critically ill patients. Given the high mortality associated with delayed IC treatment and the ineffectiveness of empiric therapy based on fever, early preemptive approaches incorporating fungal biomarkers may have a substantial advantage.
CONSORT Checklist.
(DOCX)
Trial Protocol.
(DOCX)