Comments
Abstract
Rationale: Invasive pulmonary aspergillosis has been increasingly reported in nonneutropenic patients, including those with underlying respiratory diseases.
Objectives: We compared the diagnostic performances of galactomannan, 1,3-β-D-glucan, and Aspergillus-specific lateral-flow device tests with that of conventional culture by using bronchoalveolar lavage fluid samples from patients with underlying respiratory diseases.
Methods: We analyzed 268 bronchoalveolar lavage samples from 221 patients with underlying respiratory diseases (and without hematologic malignancy or previous solid organ transplantation) that were collected for routine microbiological workup between February 2012 and May 2014 at the University Hospital of Graz, Austria. Invasive pulmonary aspergillosis was defined according to European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria modified for patients with respiratory diseases.
Measurements and Main Results: Thirty-one patients (14%) had probable or proven, 25 possible, and the remaining 165 patients no invasive pulmonary aspergillosis. Probable/proven aspergillosis was associated with a significantly higher (P = 0.034) 30-day mortality rate of 32%. Sensitivities, specificities, and diagnostic odd ratios differed markedly between galactomannan (cut-off 0.5: optical density index, 0.97, 0.81, 124.4; cut-off 1.0: 0.97, 0.93, 422.1; cut-off 3.0: 0.61, 0.99, 109.8), β-D-glucan (cut-off 80 pg/ml: 0.90, 0.42, 6.57; cut-off 200 pg/ml: 0.70, 0.61, 3.7), lateral-flow device tests (0.77, 0.92, 41.8), and mycological culture (0.29, 0.97, 14).
Conclusions: Probable or proven invasive pulmonary aspergillosis was diagnosed in 14% of our study population and associated with significantly higher 30-day mortality rates. Although the performance of β-D-glucan was limited by low specificity and that of mycological culture by low sensitivity, the Aspergillus lateral-flow device seems to be a promising alternative to galactomannan testing, which remains the diagnostic gold standard for aspergillosis.
Clinical trial registered with www.clinicaltrials.gov (NCT 02058316).
Invasive pulmonary aspergillosis (IPA) has been increasingly reported in nonneutropenic patients, including those with underlying respiratory diseases. The performance of a newly available test (the Aspergillus-specific lateral-flow device [LFD]) for diagnosis of IPA using bronchoalveolar lavage fluid has not been studied in these patients.
This study supports the clinical use of the Aspergillus LFD as an adjunct test for IPA alongside the galactomannan enzyme-immunoassay, the current gold standard test for disease detection. The Aspergillus LFD allows rapid and accurate diagnosis of the disease, facilitating earlier initiation of appropriate antifungal therapy and aiding clinical practice in the diagnosis and management of IPA in patients with underlying respiratory diseases.
Invasive pulmonary aspergillosis (IPA) is a life-threatening disease caused by the fungus Aspergillus fumigatus and other Aspergillus species and represents the leading cause of invasive fungal infection (IFI)-related morbidity and mortality in patients with hematological malignancies (1–4). Over recent years, IPA has been increasingly reported in nonneutropenic patients, including those with underlying diseases of the lung (e.g., steroid-treated chronic obstructive pulmonary disease [COPD], asthma, lung cancer, or autoimmune diseases with pulmonary involvement) (4–7). Early recognition and appropriate treatment of IPA in patients with COPD is crucial, as mortality rates greater than 90% have been reported (8), and early initiation of systemic antifungal therapy may lead to improved survival rates of up to 80% (9, 10). Diagnosis of IPA in patients with underlying respiratory diseases is difficult, however, as clinical presentation is typically characterized by nonspecific symptoms such as dyspnea, cough, or hemoptysis, and early radiological signs are also unspecific. Typical radiological signs for IPA (e.g., halo or air-crescent sign) are particularly rare in early stages of the disease in nonneutropenic patients (8, 11). Mycological culture—the previous gold standard for IPA diagnosis—is limited by low sensitivity and long turnaround time (10). Therefore, diagnostic tools based on fungal antigen detection have been developed within the last decade and have demonstrated their huge potential in IPA diagnosis (1, 12).
Galactomannan (GM) is a circulating polysaccharide component of Aspergillus species that is released into the bloodstream by growing hyphae and germinating conidia and can be detected by using a commercial ELISA (Bio-Rad Laboratories, Marnes-la-Coquette, France) (13–15). Although the performance of serum GM testing seems to be promising only in neutropenic patients, who usually develop angioinvasive forms of IPA (1, 16), bronchoalveolar lavage (BAL) fluid (BALF) GM seems the method of choice in nonneutropenic patients who tend to develop airway-invasive forms (17–19). This was confirmed in a study evaluating patients with underlying structural damage of the lung tissue (e.g., COPD, emphysema, bronchiectasis), which found that only one out of 65 patients with COPD with IPA had a positive serum GM test (8). In contrast, GM detection in BAL specimens has been reported to reach sensitivities of about 90% and specificities of 95% (18, 20–22). Limitations of GM testing include potential false-positive test results and the variable turnaround time, with time to results of 3 days or more in many centers (23–25).
These limitations may be overcome by the Aspergillus lateral-flow device (LFD) test, a point-of-care test for IPA. The LFD is an immunochromatographic assay using a monoclonal JF5 antibody to detect an extracellular antigen secreted by Aspergillus species exclusively during active growth (26). The LFD is easily performed using native BALF and yields results in 15 minutes. In recent studies, the high potential of this new assay in BAL IPA detection was shown in hematological malignancy and solid-organ transplant (SOT) patients (26–31). However, no data on the performance of the LFD test in nonneutropenic patients with underlying respiratory diseases have been published to date.
The 1,3-β-d-glucan (BDG) test is a colorimetric “panfungal” diagnostic assay that was included into mycological criteria in the revised European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria (32). BDG is a cell wall component of most pathogenic fungi, including Aspergillus, Pneumocystis and Candida species, but is not present in the cell walls of the Mucorales. The high negative predictive value (NPV) of approximately 95% (33), when performed with serum specimens, is of particular interest for clinicians to rule out IFIs. In contrast, limited data exist on its diagnostic value in BAL specimens (31, 34).
The aim of this study was to evaluate the diagnostic performances of GM, BDG, and the Aspergillus LFD for BAL IPA detection in patients with underlying respiratory disease and to compare these to conventional culture.
Some of the results of these studies have been previously reported in the form of an abstract (35).
This part-prospective (GM, LFD testing, mycological culture), part-retrospective (BDG testing) cohort study was conducted between February 2012 and May 2014 at the University Hospital of Graz, Graz, Austria. Patients older than 18 years of age with underlying respiratory diseases who underwent routine bronchoscopy and had samples sent for microbiological workup were included. Exclusion criteria were underlying hematological malignancies or previous receipt of SOT. Overall, 268 BAL samples (defined as cases) from 221 patients with underlying respiratory diseases were included. In patients for whom more than one sample was included, time between consecutive sampling was 4 days or more.
IPA was classified according to a slightly modified version of the European Organization of Research and Treatment of Cancer/Mycoses Study Group revised definitions of 2008 that includes modified mycological criteria (single BAL GM > 1.0 optical-density index [ODI] was added) and host factors (preexisting respiratory disease was added). Modifications were necessary because (1) publications about the superiority of BAL GM when compared with serum GM were performed after 2008 (20), and (2) host factors in these guidelines were originally defined for patients with hematologic malignancies and for severely immunocompromised patients (32, 36), whereas they have not been evaluated in other nonneutropenic patients at risk for IPA.
GM, LFD testing, and conventional culture were performed for all samples and always immediately after sample collection. All samples were then stored at −80°C for retrospective BDG testing (all performed within 6 mo after sample collection).
Mycological cultures and GM testing (by using the commercially available Platelia Aspergillus EIA/Ag assay [Bio-Rad, Munich, Germany]) were performed at the Institute of Hygiene, Microbiology, and Environmental Medicine, Medical University of Graz, as described previously (1). LFD testing was performed prospectively at the Microbiology Laboratory, University Hospital of Graz, according to the method described elsewhere (28).
BDG testing was performed at the Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, using the commercially available Fungitell assay (Associates of Cape Cod, Falmouth, MA) adapted for automation on a BCS-XP coagulation analyzer allowing single-sample testing with fast turnaround time, as described previously (37). BDG testing did not yield results in 14 of 268 (5%) samples due to significant hemolysis. BDG results were therefore available for 254 samples.
For statistical analysis, SPSS 21 (SPSS Inc., Chicago, IL) was used. We calculated sensitivity, specificity, NPV, positive predictive value (PPV), and diagnostic odds ratio (DOR), including 95% confidence intervals (CIs), to compare the diagnostic performance among GM, BDG, LFD, and conventional culture. Receiver operating characteristics analysis was performed and area under the curve (AUC) values (including 95% CI) displayed for GM and BDG values. For all these tests, we performed three calculations to compare (1) proven/probable IPA versus no IPA, (2) proven/probable IPA versus possible/no IPA, and (3) proven/probable/possible IPA versus no IPA.
Our study was conducted in accordance with the Declaration of Helsinki, 1996, Good Clinical Practice, and applicable local regulatory requirements and law. The study protocol was approved by the local ethics committee, Medical University Graz, Austria (EC-number 25-221 ex 12/13) and registered at ClinicalTrials.gov (identifier NCT02058316).
Demographic characteristics and underlying respiratory diseases of the study population are shown in Table 1. The majority of patients were on broad-spectrum antibiotics, and eight patients were on mold-active empirical antifungal therapy at the time bronchoscopy was performed. In six of eight patients, empirical therapy was initiated within 24 hours before bronchoscopy.
| Overall Study Population | General Ward | RCU | ICU | Patients with No IPA or Possible IPA | Patients with Probable or Proven IPA | P Value* | |
|---|---|---|---|---|---|---|---|
| No. of patients | 221 | 126 | 20 | 75 | 190 | 31 | |
| Sex | |||||||
| Male | 128 (58) | 68 (54) | 13 (65) | 47 (63) | 110 (58) | 18 (58) | |
| Female | 93 (42) | 58 (46) | 7 (35) | 28 (37) | 80 (42) | 13 (42) | |
| Median age (range), yr | 64 (18–92) | 62 (18–92) | 69.5 (51–89) | 67 (21–82) | 63 (18–89) | 69 (24–92) | |
| Primary underlying respiratory disease per patient | |||||||
| COPD | 67 (30) | 31 (25) | 9 (45) | 27 (36) | 53 (28) | 14 (45) | |
| Bacterial pneumonia | 48 (21) | 15 (12) | 7 (35) | 26 (35) | 42 (22) | 6 (19.5) | |
| Rheumatoid/autoimmune diseases with lung involvement | 46 (21) | 37 (29) | 3 (15) | 6 (8) | 42 (22) | 4 (13) | |
| Bronchial carcinoma | 35 (16) | 31 (25) | 1 (5) | 3 (4) | 29 (15) | 6 (19.5) | |
| Viral pneumonia | 9 (4) | — | — | 9 (12) | 9 (5) | — | |
| Chronic respiratory failure | 4 (2) | 2 (2) | — | 2 (3) | 4 (2) | — | |
| Bronchial asthma | 4 (2) | 4 (3) | — | — | 4 (2) | — | |
| Bronchiectasis | 5 (2) | 5 (4) | — | — | 5 (3) | — | |
| AIDS with pneumonia | 2 (1) | 1 (1) | — | 1 (1) | 1 (0.5) | 1 (3) | |
| Pulmonary tuberculosis | 1 (1) | — | — | 1 (1) | 1 (0.5) | — | |
| Care per patient | |||||||
| General ward | 126 (57) | — | — | — | 113 (60) | 13 (42) | |
| RCU | 20 (9) | — | — | — | 14 (7) | 6 (19) | 0.031 |
| ICU | 75 (34) | — | — | — | 63 (33) | 12 (39) | |
| Mortality | |||||||
| 30-d mortality | 35 (16) | 4 (3) | 5 (25) | 26 (35) | 30 (16) | 10 (32) | 0.027 |
| 90-d mortality | 48 (22) | 8 (6) | 7 (35) | 33 (44) | 40 (21) | 12 (39) | 0.031 |
| No. of cases (i.e., samples) | 268 | 129 | 35 | 104 | 237 | 31 | |
| Sex | |||||||
| Male | 149 (44) | 70 (54) | 21 (60) | 58 (56) | 131 (55) | 18 (58) | |
| Female | 119 (56) | 59 (46) | 14 (40) | 46 (44) | 106 (45) | 13 (42) | |
| Median age (range), yr | 65 (18–92) | 62 (18–92) | 69 (51–89) | 67.5 (21–82) | 64 (18–92) | 69 (24–92) | |
| Primary underlying respiratory disease at the time of BAL sampling | |||||||
| COPD | 83 (31) | 32 (25) | 15 (43) | 36 (35) | 69 (29) | 14 (45) | |
| Bacterial pneumonia | 63 (24) | 15 (12) | 12 (34) | 36 (35) | 57 (24) | 6 (19) | |
| Rheumatoid/autoimmune diseases with lung involvement | 51 (19) | 38 (29) | 4 (11) | 9 (9) | 47 (19) | 4 (13) | |
| Bronchial carcinoma | 36 (13) | 32 (25) | 1 (3) | 3 (3) | 30 (13) | 6 (19) | |
| Viral pneumonia | 15 (6) | — | — | 15 (14) | 15 (6) | — | |
| Chronic respiratory failure | 7 (3) | 2 (2) | 3 (9) | 2 (2) | 7 (3) | — | |
| Bronchial asthma | 5 (2) | 4 (3) | — | 1 (1) | 5 (2) | — | |
| Bronchiectasis | 5 (2) | 5 (4) | — | — | 5 (2) | — | |
| AIDS with pneumonia | 2 (1) | 1 (1) | — | 1 (1) | 1 (0.4) | 1 (3) | |
| Pulmonary tuberculosis | 1 (0.4) | — | — | 1 (1) | 1 (0.4) | — | |
| Care at the time of bronchoscopy | |||||||
| General ward | 129 (48) | — | — | — | 116 (49) | 13 (42) | |
| RCU | 35 (13) | — | — | — | 29 (12) | 6 (19) | |
| ICU | 104 (39) | — | — | — | 92 (39) | 12 (39) | |
| Mortality | |||||||
| 30-d mortality | 50 (19) | 4 (3) | 8 (23) | 38 (37) | 40 (17) | 10 (32) | 0.039 |
| 90-d mortality | 69 (26) | 9 (7) | 10 (29) | 50 (48) | 57 (24) | 12 (39) |
A total of 4 patients (4 cases) had proven IPA, 27 patients (27 cases) had probable IPA, 25 patients (26 cases) had possible IPA, and the remaining 165 patients (211 cases) had no evidence for IPA. Overall, 14% of patients (11.5% of cases) had probable or proven IPA. COPD was the most common underlying respiratory disease among patients with proven or probable IPA, and COPD had the highest incidence rate, as 20.9% (14 of 67 patients) of patients with COPD undergoing diagnostic bronchoscopy were diagnosed with proven/probable IPA. High rates of IPA were also found among patients with lung carcinoma (6 of 35 patients; 17.1%). Some 41.9% (13 of 31) of patients with proven/probable IPA were treated at general wards at the time of BAL sampling and 58.1% (18 of 31) at the respiratory care unit (RCU) or intensive care units (ICUs). A trend, although not significant, toward higher incidence rate of proven/probable IPA was found in patients from RCU/ICU (18/95; 18.9%) compared with patients from general wards (13/126; 10.3%, P = 0.067). Detailed results for cases treated at general wards, RCU, or ICU at the time of bronchoscopy as well as patients with and without proven/probable IPA are shown in Table 1.
Thirty- and 90-day mortality for patients with probable or proven IPA were 32% (10 of 31 patients) and 38.7% (12 of 31 patients), respectively. For possible IPA, 16% of patients died within 30 days (4 of 25 patients [5 of 26 cases]), and 28% died within 90 days (7 of 25 patients [8 of 26 cases]). For patients with no evidence for IPA, 16% of patients died within 30 days (26 of 165 patients [35 of 211 cases]) and 21% within 90 days (35 of 165 [49 of 211 cases]). The 30-day mortality rates were significantly lower both for patients (P = 0.027) and cases (P = 0.039) with possible/no IPA versus those with proven/probable IPA. Also significantly lower 90-day mortality was found for patients with possible/no IPA versus those with proven/probable (P = 0.031). No significant difference in 90-day mortality was observed for patients with proven/probable IPA treated on ICUs, RCUs, or general wards (P = 0.588).
Sensitivity, specificity, NPV, PPV, and DOR plus 95% CI for proven/probable IPA versus no IPA are shown in Table 2. Results for proven/probable IPA versus possible/no IPA are shown in Table 3, and for possible/probable and proven IPA versus no IPA in Table 4.
| Test Methods | Sensitivity | Specificity | PPV | NPV | DOR (95% CI) |
|---|---|---|---|---|---|
| BAL GM > 0.5 ODI | 97 (30/31) | 81 (170/211) | 42 (30/71) | 99 (170/171) | 124.4 (16.5–938.9) |
| BAL GM > 1.0 ODI | 97 (30/31) | 93 (197/211) | 68 (30/44) | 99 (197/198) | 422.1 (53.5–3,328) |
| BAL GM > 3.0 ODI | 61 (19/31) | 99 (208/211) | 86 (19/22) | 95 (208/220) | 109.8 (28.5–423.3) |
| BDG > 80 pg/ml | 90 (27/30) | 42 (84/199) | 19 (27/142) | 96 (84/87) | 6.6 (1.9–22.4) |
| BDG > 200 pg/ml | 70 (21/30) | 61 (122/199) | 21 (21/98) | 93 (122/131) | 3.7 (1.6–8.5) |
| LFD | 77 (24/31) | 92 (195/211) | 60 (24/40) | 97 (195/202) | 41.8 (15.6–111.8) |
| Conventional culture | 29 (9/31) | 97 (205/211) | 60 (9/15) | 90 (205/227) | 14 (4.5–43) |
| BAL GM > 3.0 ODI and/or positive LFD | 100 (31/31) | 91 (192/211) | 62 (31/50) | 100 (192/192) | 621.9 (36.6–10,563) |
| Test Methods | Sensitivity | Specificity | PPV | NPV | DOR (95% CI) |
|---|---|---|---|---|---|
| BAL GM > 0.5 ODI | 97 (30/31) | 78 (184/237) | 36 (30/83) | 99 (184/185) | 104.5 (13.9–781.7) |
| BAL GM > 1.0 ODI | 97 (30/31) | 94 (222/237) | 66 (30/45) | 99 (222/223) | 444 (56.6–3,483) |
| BAL GM > 3.0 ODI | 61 (19/31) | 99 (234/237) | 86 (19/22) | 95 (234/246) | 123.5 (33.1–475.8) |
| BDG > 80 pg/ml | 90 (27/30) | 42 (93/224) | 17 (27/158) | 97 (93/96) | 6.4 (1.9–21.7) |
| BDG > 200 pg/ml | 70 (21/30) | 59 (133/224) | 19 (21/112) | 94 (133/142) | 3.4 (1.5–7.8) |
| LFD | 77 (24/31) | 85 (202/237) | 41 (24/59) | 97 (202/209) | 19.8 (7.9–49.4) |
| Conventional culture | 29 (9/31) | 97 (231/237) | 60 (9/15) | 91 (231/253) | 15.8 (5.1–48.4) |
| BAL GM > 3.0 ODI and/or positive LFD | 100 (31/31) | 84 (199/237) | 45 (31/69) | 100 (199/199) | 326.5 (19.6–5,459) |
| Test Methods | Sensitivity | Specificity | PPV | NPV | DOR (95% CI) |
|---|---|---|---|---|---|
| BAL GM > 0.5 ODI | 72 (41/57) | 80 (169/211) | 49 (41/83) | 91 (169/185) | 10.3 (5.3–20.1) |
| BAL GM > 1.0 ODI | 53 (30/57) | 93 (196/211) | 66 (30/45) | 88 (196/223) | 14.5 (6.9–30.4) |
| BAL GM > 3.0 ODI | 33 (19/57) | 99 (208/211) | 86 (19/22) | 88 (208/246) | 34.7 (9.8–122.9) |
| BDG > 80 pg/mL | 78 (43/55) | 42 (84/199) | 27 (43/158) | 88 (84/96) | 6.4 (1.9–21.7) |
| BDG > 200 pg/mL | 64 (35/55) | 61 (122/199) | 29 (35/112) | 86 (122/142) | 2.8 (1.5–5.1) |
| LFD | 75 (43/57) | 92 (195/211) | 73 (43/59) | 93 (195/209) | 37.4 (17–82.4) |
| Conventional Culture | 16 (9/57) | 97 (205/211) | 60 (9/15) | 81 (205/253) | 6.4 (2.2–18.9) |
| BAL GM > 3.0 ODI and/or positive LFD | 88 (50/57) | 91 (192/211) | 72 (50/69) | 96 (192/199) | 72.2 (28.7–181.3) |
Receiver operating characteristics analysis revealed an AUC value of 0.965 (95% CI, 0.935–0.996) for GM and 0.752 (95% CI, 0.662–0.842) for BDG for differentiation between proven/probable IPA versus no IPA (Figure 1). For differentiation between proven/probable IPA versus possible/no IPA, AUCs were as follows: GM 0.966 (95% CI, 0.934–0.998), BDG 0.743 (95% CI, 0.652–0.833). For differentiation of proven/probable/possible IPA versus no IPA, GM AUC was 0.819 (95% CI, 0.746–0.893) and BDG AUC was 0.676 (95% CI, 0.594–0.758).
Figure 1. Receiver operating characteristics curve analysis for proven/probable invasive pulmonary aspergillosis (IPA) versus no IPA. BDG = 1,3-β-d-glucan; GM = galactomannan.
[More] [Minimize]GM levels between 0.5 and 1.0 ODI were found in 11 of 26 (42.3%) cases with possible IPA and 27 of 211 (13%) cases without evidence for IPA.
In 68 patients (81 cases), BAL cultures grew Candida species and all were interpreted as colonization. Ninety-three percent of samples (75 of 81 cases) had BDG levels greater than 80 pg/ml, and 72% (58 of 81 samples) had BDG levels greater than 200 pg/ml. Forty-four of these 81 (54.3%) cases were hospitalized at ICU, 14 (17.3%) cases at RCU, and 23 (28.4%) on general wards. In one patient, BAL culture grew Penicillium species (another mold), but GM, BDG, and LFD remained negative.
We assessed the diagnostic performance of GM, BDG, and the Aspergillus LFD test as well as conventional culture for IPA in BAL samples in a cohort of patients with underlying respiratory disease. There were three major findings. First, proven or probable IPA was diagnosed in 14% of patients with underlying respiratory diseases where BAL was sent for microbiological workup and was associated with significantly higher 30- and 90-day mortality rates. Second, Aspergillus LFD testing of BAL specimens seems a promising tool for IPA diagnosis, especially if GM results are not rapidly available. Third, performances of BDG (specificity < 50%) and conventional culture (sensitivity around 30%) were less convincing.
COPD was the most frequently observed underlying respiratory disease in both our overall study population (30%) and those with proven/probable IPA (45%). Patients with COPD undergoing bronchoscopy also had the highest IPA rate (20.9%), followed by patients with lung cancer (17.1%). Previously reported IPA incidence in patients with chronic lung diseases varies widely from 1.9% in patients with acute exacerbation of COPD (38) to 22% in patients with COPD with isolation of Aspergillus species from the upper respiratory tract (7). In patients with underlying respiratory diseases, patients with COPD seem to be most vulnerable for IPA development (7, 39–42). A possible explanation is found in pathophysiology. COPD leads to various changes in the local immune response to Aspergillus species. Impairment of polymorphonuclear neutrophils, eosinophils, macrophages, lymphocytes, as well as a decrease of phagocytosis and antigen recognition in lung tissues may result in a shift from fungal colonization to fungal infection (39, 43, 44). This study also found a high IPA rate among patients with lung cancer undergoing bronchoscopy with microbiological work up. Reported IPA incidence rates in patients with bronchial carcinoma vary widely between 2.6 and 40.6% (6, 45) and mortality reaches 71.5% (5). High-dose systemic corticosteroid treatment in cases of central nervous system metastasis, tumor necrosis, poststenotic mucus retention, as well as advanced stage of bronchial carcinoma and recent chemotherapy have been described as the main risk factors in those patients (5, 6, 45).
Overall, the 30-day mortality rate for patients with proven or probable IPA was 32% in this study cohort and was therefore comparable to previously published mortality rates in patients with hematological malignancies of 34% (3).
We also determined that a BAL GM cut-off of 1.0 ODI was the most promising threshold value for IPA detection in this nonneutropenic cohort without antifungal prophylaxis, with a sensitivity of 97% and specificity of 93% for proven/probable IPA compared with no IPA. Lowering the cut-off value to 0.5 ODI would have resulted in a lower test specificity of 81% (i.e., one out of five patients without IPA would have had a false-positive GM result) but also an increase of probable IPA cases (in this study, 42% of patients with possible IPA had GM results between 0.5 and 1.0 ODI). Increasing the cut-off for GM in BAL samples to an ODI of 3.0 would have led to a decrease of sensitivity (when compared with culture results) and a slight increase in test specificity. Combining GM with a cut-off of 3.0 with LFD was, however, promising.
Because GM testing can be limited by a long diagnostic turnaround time, the Aspergillus LFD point-of-care test may represent a promising alternative for the rapid and reliable diagnosis of IPA with BAL specimens. This is the first study to investigate the diagnostic performance of the BAL LFD test in patients with underlying respiratory diseases and without hematological malignancies or previous SOT. Previous studies evaluated the diagnostic performance of BAL LFD test in different patient populations: in SOT recipients sensitivity reached 91% and specificity 88% (29), and in patients with underlying hematological malignancies sensitivity reached 85% and specificity 100% when compared with patients without IPA (27, 31). In this study, the sensitivity of the LFD for IPA diagnosis in patients with underlying respiratory diseases was 77%, and the specificity was 92%, for proven/probable disease (vs. no IPA) and was therefore comparable to previous results in other patient populations, indicating the high diagnostic potential of this test when combined with BAL samples. Of interest is also the high NPV of the BAL LFD test, which was 97% in this study and also between 95 and 100% in previous studies (27, 29, 31). Azoulay and colleagues previously reported that up to two-thirds of patients receiving antifungal therapy in the ICU setting have no evidence for IFIs (46). Overtreatment with antifungals is not only associated with a huge financial burden but also bears the risk of side effects, drug–drug interactions, and even development of resistance (47, 48). Thus, using the LFD test may be an effective way to reduce unnecessary antifungal treatment.
The BDG assay, when tested in BAL, was limited by a low specificity varying from 42 to 61%. Even increasing the cut-off from 80 pg/ml to 200 pg/ml did not lead to a definite increase in specificity. The high frequency of positive BDG results with BAL specimen is not surprising, however, as BDG is also produced by Candida species, and colonization of the respiratory tract by Candida species occurs frequently, whereas pneumonia due to Candida species is an absolute rarity (49). In this study, the BDG test resulted positive in 93 and 72% of patients with Candida colonization, when detection thresholds of 80 pg/ml and 200 pg/ml were used, respectively. Although specificity has been described as the major limitation of the BDG assay in recent smaller studies, specificity was markedly lower in this study (31, 34). Differences in specificity (a previous study reported a specificity of 76% for the 80 pg/ml cut-off) may be explained by the fact that different proportions of the study populations were treated at ICUs, which is associated with higher Candida colonization rates of the respiratory tract (49). In this study, 38.8% of all patients were treated at ICUs, whereas only 6% of patients in the previous study required ICU treatment (31). Potential false positivity due to Candida colonization is therefore a major limiting factor for BDG diagnosis of IPA when using BAL. However, the high NPV (up to 97% using the 80 pg/ml cut-off) may be useful to rule out clinical suspicion of IPA.
In this study, we observed a low sensitivity (29%) of mycological culture for proven/probable IPA, with a PPV of 60%. This is in accordance with previously published data reporting sensitivities of 30% and less (10). However, there are several factors warranting the use of mycological culture. First, culture is necessary for susceptibility testing, which is of vital importance because azole resistance in Aspergillus species has emerged (50). Second, culture may also detect other molds (e.g., Mucorales) that give negative biomarker results.
Our study has several limitations, including its single-center design, which meant that numbers, particular those of proven IPA cases, were low. Another limitation is the absence of reliable definitions of possible and probable IPA in nonneutropenic patients, which necessitated modifications to the definitions used for neutropenic patients. Retrospective BDG testing may also be considered another limitation of our study, as it was reported recently that reproducibility of BDG evaluations using frozen BAL samples may be poor, although no trends in sensitivity have been found (34). Finally, only patients in whom BAL was routinely obtained and sent for microbiological workup were included, which likely resulted in an overestimation of IPA rates.
In summary, the GM-ELISA and Aspergillus LFD showed the best diagnostic performance for IPA diagnosis when used with BAL specimens in a cohort of nonneutropenic patients with underlying respiratory diseases. The performance of the BDG test was limited by low specificity and that of mycological culture by low sensitivity. Our results show that the Aspergillus LFD test may be a promising alternative to the GM test, which is the current the gold standard for IPA diagnosis.
| 1. | Hoenigl M, Salzer HJ, Raggam RB, Valentin T, Rohn A, Woelfler A, Seeber K, Linkesch W, Krause R. Impact of galactomannan testing on the prevalence of invasive aspergillosis in patients with hematological malignancies. Med Mycol 2012;50:266–269. |
| 2. | Hoenigl M, Zollner-Schwetz I, Sill H, Linkesch W, Lass-Flörl C, Schnedl WJ, Krause R. Epidemiology of invasive fungal infections and rationale for antifungal therapy in patients with haematological malignancies. Mycoses 2011;54:454–459. |
| 3. | Perkhofer S, Lass-Flörl C, Hell M, Russ G, Krause R, Hönigl M, Geltner C, Auberger J, Gastl G, Mitterbauer M, et al. The Nationwide Austrian Aspergillus Registry: a prospective data collection on epidemiology, therapy and outcome of invasive mould infections in immunocompromised and/or immunosuppressed patients. Int J Antimicrob Agents 2010;36:531–536. |
| 4. | Cornillet A, Camus C, Nimubona S, Gandemer V, Tattevin P, Belleguic C, Chevrier S, Meunier C, Lebert C, Aupée M, et al. Comparison of epidemiological, clinical, and biological features of invasive aspergillosis in neutropenic and nonneutropenic patients: a 6-year survey. Clin Infect Dis 2006;43:577–584. |
| 5. | Peghin M, Ruiz-Camps I, Garcia-Vidal C, Cervera C, Andreu J, Martin M, Gavaldá J, Gudiol C, Moreno A, Felip E, et al. Unusual forms of subacute invasive pulmonary aspergillosis in patients with solid tumors. J Infect 2014;69:387–395. |
| 6. | Yan X, Li M, Jiang M, Zou LQ, Luo F, Jiang Y. Clinical characteristics of 45 patients with invasive pulmonary aspergillosis: retrospective analysis of 1711 lung cancer cases. Cancer 2009;115:5018–5025. |
| 7. | Guinea J, Torres-Narbona M, Gijón P, Muñoz P, Pozo F, Peláez T, de Miguel J, Bouza E. Pulmonary aspergillosis in patients with chronic obstructive pulmonary disease: incidence, risk factors, and outcome. Clin Microbiol Infect 2010;16:870–877. |
| 8. | Samarakoon P, Soubani A. Invasive pulmonary aspergillosis in patients with COPD: a report of five cases and systematic review of the literature. Chron Respir Dis 2008;5:19–27. |
| 9. | Greene RE, Schlamm HT, Oestmann JW, Stark P, Durand C, Lortholary O, Wingard JR, Herbrecht R, Ribaud P, Patterson TF, et al. Imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign. Clin Infect Dis 2007;44:373–379. |
| 10. | Lass-Flörl C, Resch G, Nachbaur D, Mayr A, Gastl G, Auberger J, Bialek R, Freund MC. The value of computed tomography-guided percutaneous lung biopsy for diagnosis of invasive fungal infection in immunocompromised patients. Clin Infect Dis 2007;45:e101–e104. |
| 11. | Nucci M, Nouér SA, Grazziutti M, Kumar NS, Barlogie B, Anaissie E. Probable invasive aspergillosis without prespecified radiologic findings: proposal for inclusion of a new category of aspergillosis and implications for studying novel therapies. Clin Infect Dis 2010;51:1273–1280. |
| 12. | Hoenigl M, Valentin T, Salzer HJ, Zollner-Schwetz I, Krause R. Underestimating the real burden of invasive fungal infections in hematopoietic stem cell transplant recipients? Clin Infect Dis 2010;51:253–254, author reply 254–255. |
| 13. | Maertens J, Maertens V, Theunissen K, Meersseman W, Meersseman P, Meers S, Verbeken E, Verhoef G, Van Eldere J, Lagrou K. Bronchoalveolar lavage fluid galactomannan for the diagnosis of invasive pulmonary aspergillosis in patients with hematologic diseases. Clin Infect Dis 2009;49:1688–1693. |
| 14. | Marr KA, Balajee SA, McLaughlin L, Tabouret M, Bentsen C, Walsh TJ. Detection of galactomannan antigenemia by enzyme immunoassay for the diagnosis of invasive aspergillosis: variables that affect performance. J Infect Dis 2004;190:641–649. |
| 15. | Wheat LJ, Nguyen MH, Alexander BD, Denning D, Caliendo AM, Lyon GM, Baden LR, Marty FM, Clancy C, Kirsch E, et al. Long-term stability at -20 °C of Aspergillus galactomannan in serum and bronchoalveolar lavage specimens. J Clin Microbiol 2014;52:2108–2111. |
| 16. | Husain S, Kwak EJ, Obman A, Wagener MM, Kusne S, Stout JE, McCurry KR, Singh N. Prospective assessment of Platelia Aspergillus galactomannan antigen for the diagnosis of invasive aspergillosis in lung transplant recipients. Am J Transplant 2004;4:796–802. |
| 17. | Cordonnier C, Botterel F, Ben Amor R, Pautas C, Maury S, Kuentz M, Hicheri Y, Bastuji-Garin S, Bretagne S. Correlation between galactomannan antigen levels in serum and neutrophil counts in haematological patients with invasive aspergillosis. Clin Microbiol Infect 2009;15:81–86. |
| 18. | Meersseman W, Lagrou K, Maertens J, Wilmer A, Hermans G, Vanderschueren S, Spriet I, Verbeken E, Van Wijngaerden E. Galactomannan in bronchoalveolar lavage fluid: a tool for diagnosing aspergillosis in intensive care unit patients. Am J Respir Crit Care Med 2008;177:27–34. |
| 19. | Bergeron A, Porcher R, Sulahian A, de Bazelaire C, Chagnon K, Raffoux E, Vekhoff A, Cornet M, Isnard F, Brethon B, et al. The strategy for the diagnosis of invasive pulmonary aspergillosis should depend on both the underlying condition and the leukocyte count of patients with hematologic malignancies. Blood 2012;119:1831–1837; quiz 1956. |
| 20. | Zou M, Tang L, Zhao S, Zhao Z, Chen L, Chen P, Huang Z, Li J, Chen L, Fan X. Systematic review and meta-analysis of detecting galactomannan in bronchoalveolar lavage fluid for diagnosing invasive aspergillosis. PLoS ONE 2012;7:e43347. |
| 21. | Buchheidt D, Hummel M, Schleiermacher D, Spiess B, Schwerdtfeger R, Cornely OA, Wilhelm S, Reuter S, Kern W, Südhoff T, et al. Prospective clinical evaluation of a LightCycler-mediated polymerase chain reaction assay, a nested-PCR assay and a galactomannan enzyme-linked immunosorbent assay for detection of invasive aspergillosis in neutropenic cancer patients and haematological stem cell transplant recipients. Br J Haematol 2004;125:196–202. |
| 22. | D’Haese J, Theunissen K, Vermeulen E, Schoemans H, De Vlieger G, Lammertijn L, Meersseman P, Meersseman W, Lagrou K, Maertens J. Detection of galactomannan in bronchoalveolar lavage fluid samples of patients at risk for invasive pulmonary aspergillosis: analytical and clinical validity. J Clin Microbiol 2012;50:1258–1263. |
| 23. | Marr KA, Laverdiere M, Gugel A, Leisenring W. Antifungal therapy decreases sensitivity of the Aspergillus galactomannan enzyme immunoassay. Clin Infect Dis 2005;40:1762–1769. |
| 24. | Reinwald M, Hummel M, Kovalevskaya E, Spiess B, Heinz WJ, Vehreschild JJ, Schultheis B, Krause SW, Claus B, Suedhoff T, et al. Therapy with antifungals decreases the diagnostic performance of PCR for diagnosing invasive aspergillosis in bronchoalveolar lavage samples of patients with haematological malignancies. J Antimicrob Chemother 2012;67:2260–2267. |
| 25. | Affolter K, Tamm M, Jahn K, Halter J, Passweg J, Hirsch HH, Stolz D. Galactomannan in bronchoalveolar lavage for diagnosing invasive fungal disease. Am J Respir Crit Care Med 2014;190:309–317. |
| 26. | Thornton CR. Development of an immunochromatographic lateral-flow device for rapid serodiagnosis of invasive aspergillosis. Clin Vaccine Immunol 2008;15:1095–1105. |
| 27. | Hoenigl M, Koidl C, Duettmann W, Seeber K, Wagner J, Buzina W, Wölfler A, Raggam RB, Thornton CR, Krause R. Bronchoalveolar lavage lateral-flow device test for invasive pulmonary aspergillosis diagnosis in haematological malignancy and solid organ transplant patients. J Infect 2012;65:588–591. |
| 28. | Thornton C, Johnson G, Agrawal S. Detection of invasive pulmonary aspergillosis in haematological malignancy patients by using lateral-flow technology. J Vis Exp 2012;(61):3721. |
| 29. | Willinger B, Lackner M, Lass-Flörl C, Prattes J, Posch V, Selitsch B, Eschertzhuber S, Hönigl K, Koidl C, Sereinigg M, et al. Bronchoalveolar lavage lateral-flow device test for invasive pulmonary aspergillosis in solid organ transplant patients: a semiprospective multicenter study. Transplantation (In press) |
| 30. | Wiederhold NP, Najvar LK, Bocanegra R, Kirkpatrick WR, Patterson TF, Thornton CR. Interlaboratory and interstudy reproducibility of a novel lateral-flow device and influence of antifungal therapy on detection of invasive pulmonary aspergillosis. J Clin Microbiol 2013;51:459–465. |
| 31. | Hoenigl M, Prattes J, Spiess B, Wagner J, Prueller F, Raggam RB, Posch V, Duettmann W, Hoenigl K, Wölfler A, et al. Performance of galactomannan, beta-d-glucan, Aspergillus lateral-flow device, conventional culture, and PCR tests with bronchoalveolar lavage fluid for diagnosis of invasive pulmonary aspergillosis. J Clin Microbiol 2014;52:2039–2045. |
| 32. | De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, et al.; European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 2008;46:1813–1821. |
| 33. | Lamoth F, Cruciani M, Mengoli C, Castagnola E, Lortholary O, Richardson M, Marchetti O; Third European Conference on Infections in Leukemia (ECIL-3). β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3). Clin Infect Dis 2012;54:633–643. |
| 34. | Rose SR, Vallabhajosyula S, Velez MG, Fedorko DP, VanRaden MJ, Gea-Banacloche JC, Lionakis MS. The utility of bronchoalveolar lavage beta-D-glucan testing for the diagnosis of invasive fungal infections. J Infect 2014;69:278–283. |
| 35. | Hoenigl M, Thornton C, Duettmann W, Posch V, Seeber K, Krause R, Lackner M, Lass-Flörl C. Bronchoalveolar lavage lateral-flow device test for diagnosing invasive pulmonary aspergillosis in pulmonology and ICU patients [abstract]. Am J Respir Crit Care Med 2014;189:A2695. |
| 36. | Hoenigl M, Strenger V, Buzina W, Valentin T, Koidl C, Wölfler A, Seeber K, Valentin A, Strohmeier AT, Zollner-Schwetz I, et al. European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) host factors and invasive fungal infections in patients with haematological malignancies. J Antimicrob Chemother 2012;67:2029–2033. |
| 37. | Prüller F, Wagner J, Raggam RB, Hoenigl M, Kessler HH, Truschnig-Wilders M, Krause R. Automation of serum (1→3)-beta-D-glucan testing allows reliable and rapid discrimination of patients with and without candidemia. Med Mycol 2014;52:455–461. |
| 38. | Gao X, Chen L, Hu G, Mei H. Invasive pulmonary aspergillosis in acute exacerbation of chronic obstructive pulmonary disease and the diagnostic value of combined serological tests. Ann Saudi Med 2010;30:193–197. |
| 39. | Ader F. Invasive pulmonary aspergillosis in patients with chronic obstructive pulmonary disease: an emerging fungal disease. Curr Infect Dis Rep 2010;12:409–416. |
| 40. | Bulpa PA, Dive AM, Garrino MG, Delos MA, Gonzalez MR, Evrard PA, Glupczynski Y, Installé EJ. Chronic obstructive pulmonary disease patients with invasive pulmonary aspergillosis: benefits of intensive care? Intensive Care Med 2001;27:59–67. |
| 41. | Meersseman W, Vandecasteele SJ, Wilmer A, Verbeken E, Peetermans WE, Van Wijngaerden E. Invasive aspergillosis in critically ill patients without malignancy. Am J Respir Crit Care Med 2004;170:621–625. |
| 42. | Vandewoude KH, Blot SI, Benoit D, Colardyn F, Vogelaers D. Invasive aspergillosis in critically ill patients: attributable mortality and excesses in length of ICU stay and ventilator dependence. J Hosp Infect 2004;56:269–276. |
| 43. | Berenson CS, Wrona CT, Grove LJ, Maloney J, Garlipp MA, Wallace PK, Stewart CC, Sethi S. Impaired alveolar macrophage response to Haemophilus antigens in chronic obstructive lung disease. Am J Respir Crit Care Med 2006;174:31–40. |
| 44. | Chung KF, Adcock IM. Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction. Eur Respir J 2008;31:1334–1356. |
| 45. | Shahid M, Malik A, Bhargava R. Bronchogenic carcinoma and secondary aspergillosis—common yet unexplored: evaluation of the role of bronchoalveolar lavage-polymerase chain reaction and some nonvalidated serologic methods to establish early diagnosis. Cancer 2008;113:547–558. |
| 46. | Azoulay E, Dupont H, Tabah A, Lortholary O, Stahl JP, Francais A, Martin C, Guidet B, Timsit JF. Systemic antifungal therapy in critically ill patients without invasive fungal infection. Crit Care Med 2012;40:813–822. |
| 47. | Bader O, Weig M, Reichard U, Lugert R, Kuhns M, Christner M, Held J, Peter S, Schumacher U, Buchheidt D, et al.; MykoLabNet-D Partners. cyp51A-Based mechanisms of Aspergillus fumigatus azole drug resistance present in clinical samples from Germany. Antimicrob Agents Chemother 2013;57:3513–3517. |
| 48. | Hoenigl M, Duettmann W, Raggam RB, Seeber K, Troppan K, Fruhwald S, Prueller F, Wagner J, Valentin T, Zollner-Schwetz I, et al. Potential factors for inadequate voriconazole plasma concentrations in intensive care unit patients and patients with hematological malignancies. Antimicrob Agents Chemother 2013;57:3262–3267. |
| 49. | Meersseman W, Lagrou K, Spriet I, Maertens J, Verbeken E, Peetermans WE, Van Wijngaerden E. Significance of the isolation of Candida species from airway samples in critically ill patients: a prospective, autopsy study. Intensive Care Med 2009;35:1526–1531. |
| 50. | Vermeulen E, Lagrou K, Verweij PE. Azole resistance in Aspergillus fumigatus: a growing public health concern. Curr Opin Infect Dis 2013;26:493–500. |
Funded by an investigator-initiated research grant WI174981 from Pfizer. LFD tests used in this study were provided by Dr. C. R. Thornton, University of Exeter.
Author Contributions: M.H. and J.P. designed the study. Tests were performed by J.P., F.P., C.K., S.E., W.B., and R.B.R. Data were analyzed by M.H., J.P., R.B.R., M.P., H.F., C.R.T., and R.K. Reagents, materials, and analysis tools were provided by F.P., C.K., R.B.R., I.Z.-S., W.B., and C.R.T. Samples were collected by M.P., S.E., and H.F. The manuscript was drafted by J.P., H.F., and M.H. The manuscript was critically revised and important intellectual content provided by F.P., C.K., R.B.R., M.P., S.E., H.F., I.Z.-S., C.R.T., W.B., and R.K. The final version for publication was approved by all authors. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Originally Published in Press as DOI: 10.1164/rccm.201407-1275OC on September 9, 2014
Author disclosures are available with the text of this article at www.atsjournals.org.
