ABSTRACT
The Aspergillus-specific lateral-flow device (AspLFD) test is a newly developed point-of-care diagnostic method for invasive pulmonary aspergillosis. However, evidence of the diagnostic performance of the AspLFD for chronic pulmonary aspergillosis (CPA) is limited. Therefore, we conducted a retrospective study to investigate this in comparison with the galactomannan (GM) β-d-glucan (BDG) test. Fifty patients with chronic pulmonary aspergillosis and 65 patients with respiratory disease, as a control, were enrolled in this study. The majority of the CPA disease entities were chronic pulmonary aspergillosis (64.0%, n = 32), followed by subacute invasive pulmonary aspergillosis (IPA) (20.0%, n = 10) and simple pulmonary aspergilloma (SPA) (16.0%, n = 8). The sensitivity and specificity of the AspLFD test in serum samples were 62.0% and 67.7%, respectively. The GM test (cutoff index, 1.54) showed a sensitivity of 22% and a specificity of 92.3%, while the sensitivity and specificity of the BDG test (cutoff, 19.3 pg/ml) were 48% and 90.8%, respectively. In bronchoalveolar lavage fluid samples, the AspLFD test showed a sensitivity of 66.7% and a specificity of 69.2%, while those of the GM test (cutoff index, 0.6) were 72.7% and 83.1%, respectively. The Aspergillus precipitating antibody test had 70% sensitivity. Unlike the Aspergillus precipitating antibody test, the AspLFD on serum samples showed similar sensitivity to non-fumigatus Aspergillus species. Patients with false-positive results for the AspLFD on serum samples were of a significantly higher age and had a higher prevalence of cavitary lesions in chest computed tomography than patients with negative results in the control group. Given the results in this study, the performance of the AspLFD using serum was acceptable as a point-of-care test for the diagnosis of CPA.
INTRODUCTION
Chronic pulmonary aspergillosis (CPA) is a slowly progressive disease that presents a variety of clinical courses and radiological findings. The definitive diagnosis of CPA is not easy because of the low sensitivity of mycological culture (1–4) and the difficulty of performing histopathological examination due to patients’ severe general condition. Therefore, serodiagnosis is indispensable for the diagnosis of CPA. However, the galactomannan (GM) assay does not possess high sensitivity or specificity for the diagnosis of CPA (1, 3), unlike the Aspergillus IgG precipitating antibody test (1). Aspergillus IgG antibody enzyme-linked immunosorbent assays (ELISAs) are available in several countries, and some of them are reported to show a high sensitivity and specificity for the diagnosis of CPA, though evidence for diagnosing CPA caused by non-fumigatus Aspergillus strains is lacking (5–7).
The number of CPA patients is estimated to be up to 3,000,000 per year worldwide (8); however, some CPA cases are considered to be undiagnosed in resource-limited countries, and therefore, point-of-care (POC) diagnostic methods are required (9). The diagnostic tests described above need at least half a day of turnaround time. The Aspergillus-specific lateral-flow device (AspLFD) is a newly developed POC diagnostic method for IPA. It uses the mouse monoclonal antibody JF5, which binds to a protein epitope present on an extracellular glycoprotein antigen secreted constitutively during the active growth of Aspergillus fumigatus. This test can detect Aspergillus antigens in human serum within 15 min (10). An early clinical trial showed that the AspLFD test is comparable to the GM test in serum in terms of diagnosing IPA, with sensitivity and specificity of 81.8% and 98%, respectively (11). However, evidence of the diagnostic performance of the AspLFD for CPA is limited. Therefore, we conducted a retrospective study to investigate the diagnostic performance of the AspLFD in serum and bronchoalveolar lavage (BAL) fluid in comparison with the GM assay in serum/BAL fluid and the β-d-glucan (BDG) assay in serum.
MATERIALS AND METHODS
Patients.CPA patients who were diagnosed between 2008 and 2017 and had available stored serum or bronchoalveolar lavage (BAL) fluid samples taken before antifungal treatments were enrolled in this study. Patients with respiratory diseases and without Aspergillus infection who underwent all the following tests for the diagnosis of respiratory infectious diseases in the same period were enrolled as control group patients: the required tests were bronchoscopy and serum/BAL fluid GM, BDG, and Aspergillus precipitating antibody assays. BAL was performed, using 20 to 40 ml of normal saline, at the site of the pulmonary lesion. Clinical information, including sex, age, underlying diseases, medication, laboratory test results, culture results, and chest computed tomography (CT) findings, was retrospectively collected from medical records. This study was approved by the Nagasaki University School of Medicine Research Ethics Committee (approval number 15032356-2).
Case definition.Criteria for CPA were based on the diagnostic criteria of the European Society for Clinical Microbiology and Infectious Diseases/European Respiratory Society guidelines, including (i) one or more cavities with or without a fungal ball present or nodules identified on computed tomography scan and (ii) direct evidence of Aspergillus infection or positive result of Aspergillus precipitating antibody assay (12). Patients with CPA were further classified into simple pulmonary aspergillosis (13), chronic cavitary aspergillosis (CCPA), and subacute invasive aspergillosis (SAIA) groups according to the guidelines (12).
AspLFD.The newly improved, CE-marked Aspergillus lateral flow device (AspLFD; OLM Diagnostics, Newcastle upon Tyne, United Kingdom) was used in accordance with the manufacturer’s protocol. Serum and BAL fluid samples stored at −80°C were thawed, vortexed, and centrifuged for 1 min at 14,000 × g. Serum samples were mixed with sample buffer (provided by OLM) at a ratio of 1:2 and heated in the heat block at 100°C for 3 min. Amounts of 70 μl of treated serum and untreated BAL fluid samples were applied to the port of the cassette, and the results read 15 min later. To eliminate bias, two interpreters read the AspLFD test results without knowing the diagnosis. If the two readings did not match, a third interpreter blinded to clinical information determined the results.
Galactomannan assay, β-d-glucan assay, and Aspergillus precipitating antibody.The Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Tokyo, Japan) was used for the GM assay in serum and BAL fluid samples. The FSK1 Aspergillus immunodiffusion system (Microgen Bioproducts Ltd., United Kingdom) was used for Aspergillus precipitating antibody assays in serum samples according to the manufacturer’s instructions. The Fungitec G test MK-II (Nissui, Tokyo, Japan) was used for BDG assays in plasma samples according to the manufacturer’s instructions; briefly, amounts of 5 μl of plasma samples were mixed with the pretreatment solution and heated at 37°C for 10 min in a 96-well plate. After a heating step, amounts of 100 μl of reactive solutions were added to the samples and the mixtures stirred for 60 s and then heated again at 37°C for 30 min. The absorbance was measured at 405/492 nm. All the tests were performed by technicians who were blinded to the patients’ detailed conditions.
Statistical analysis.The differences in categorical variables or continuous numbers were analyzed by Fisher’s exact test or unpaired t test. A P value of <0.05 was considered statistically significant. Receiver operating characteristic (ROC) curve analyses were performed on data from GM and BDG assays, and areas under the curves (AUCs), including 95% confidence intervals, were evaluated to assess the diagnostic ability to distinguish between CPA patients and control group patients with respiratory diseases. The Youden index was applied to determine the optimized cutoff in ROC curve analysis.
RESULTS
Patients’ characteristics.A total of 50 CPA patients and 65 disease control patients were enrolled, and the clinical characteristics of these patients are summarized in Table 1. The majority of CPA disease entities were CCPA (64%, n = 32), followed by SAIA (20%, n = 10) and SPA (16%, n = 8). Bronchoscopy was performed in 41 of 50 CPA patients, and BAL stock samples were available in 34 cases. The underlying pulmonary diseases were interstitial pneumonia, nontuberculosis mycobacterium infection, prior pulmonary tuberculosis infection, chronic obstructive pulmonary diseases, and diabetes mellitus. Steroid or immunosuppressant use was observed as a systemic immunosuppressive factor, but no patient had neutropenia. A. fumigatus (56%) was the main pathogen isolated in sputum or BAL samples; 13 cases (26%) were culture negative.
Characteristics of patients enrolled in this study and results for each test
AspLFD.The sensitivity and specificity of the Aspergillus-specific lateral-flow device (AspLFD) were 62% and 67.7%, respectively, in serum samples and 66.7% and 69.2%, respectively, in BAL fluid samples (Table 2). In serum samples, the concordance rates of the LFD test with the GM test (cutoff index, 1.54) and the BDG test (cutoff, 19.3 pg/ml) were 65.2 and 60.0%, respectively. In BAL fluid samples, the concordance rate of the LFD test with the GM test (cutoff index, 0.6) was 64.3%. Cause analysis of AspLFD false-positive cases in the control group was performed; the patients with AspLFD false-positive results were significantly older and had higher prevalences of a cavitary lesion(s) in the chest CT than the patients with negative results in the control group (Table 3). In addition, these patients showed significantly higher GM indices than patients with negative AspLFD results. The diagnostic performances for the use of AspLFD in combination with GM or BDG tests are summarized in Table 2.
Diagnostic performances of the AspLFD, GM, and BDG tests
Cause analysis of false-positive serum AspLFD results in control group
GM antigen, β-d-glucan, and Aspergillus precipitating antibody assays.The sensitivity and specificity of the serum GM antigen assay were 64% and 43.1%, respectively, at a cutoff of 0.5 and 34% and 72.3% at a cutoff of 1.0. The optimized cutoff index of this assay from the ROC curve was 1.54, with a sensitivity of 22% and a specificity of 92.3%, while the BAL fluid GM antigen assay showed higher sensitivity and specificity of 72.7% and 83.1%, respectively, at a cutoff index of 0.6 optimized from the ROC curve (Table 2). The AUCs of the ROC curves were 0.561 and 0.824, respectively (Fig. 1A and B). The serum BDG assay showed low sensitivity (40.6%) and high specificity (90.7%) at the 20-pg/ml cutoff value, which is the optimized cutoff value for Candidiasis. The optimized CPA cutoff value here was 19.3 pg/ml, with 48% sensitivity and 90.8% specificity. Its AUC for the ROC curve was 0.721, which was superior to that of the serum GM assay. The Aspergillus precipitating antibody assay showed a sensitivity of 70%.
Receiver operating characteristic curves of galactomannan (GM) antigen tests in serum and bronchoalveolar lavage (BAL) fluid and β-d-glucan test in serum. Areas under the curves (95% confidence interval) for serum GM (A), bronchoalveolar GM (B), and serum β-d-glucan test (C) were 0.561 (0.4545 to 0.6679), 0.824 (0.7322 to 0.9163), and 0.721 (0.6259 to 0.8156), respectively.
Positivity rates for non-fumigatus Aspergillus spp.The positivity rates of each test in nine CPA patients with disease caused by non-fumigatus Aspergillus spp. are summarized in Table 4. The sensitivity of the LFD for serum samples was 66.7%, similar to the result for A. fumigatus (62.9%). The BDG assay (cutoff, 19.3 pg/ml) showed results similar to those of the LFD. However, the GM assay (cutoff index, 1.54) and the Aspergillus precipitating antibody assay both showed a low sensitivity of 22.2% in this population.
Positivity rate of each test in patients with CPA caused by non-fumigatus Aspergillus spp.
DISCUSSION
In this study, the serum AspLFD assay showed better performance than the serum GM or BDG assay for the diagnosis of CPA. Moreover, its sensitivity was not inferior to that of the Aspergillus precipitating antibody assay, and unlike the Aspergillus precipitating antibody assay, it did not show any species specificity. The AspLFD also showed a high false-positive rate of 32% in the respiratory disease control group; however, since two factors, higher age and cavitary lesions, observed as false-positive factors in the cause analysis are also the common risk factors of CPA (12, 14), these cases could be undiagnosed CPA cases. The sensitivity and specificity of the AspLFD for serum samples were similar to those from a recent study in nonneutropenic invasive pulmonary aspergillosis (IPA) patients (15).
On the other hand, the diagnostic performance of the AspLFD in BAL fluid samples was similar to its performance in serum samples and, moreover, slightly inferior to that of the GM assay in BAL fluid samples. In addition, given its nature as a POC test, the utility of the AspLFD for BAL fluid samples might be limited. A recent study assessed the performance of the AspLFD in BAL fluid samples and reported that the LFD was positive in only 7% of CPA patients (16), which was an extremely low sensitivity compared to recent studies for the diagnosis of IPA (15, 17). However, the sensitivity of the BAL fluid GM index was also lower (41% at a cutoff value of 0.5) in that study (16) than in our study or other previous studies (18–20). The proportion of each disease entity of CPA in that study did not differ from those in our study. However, the volume of lavage fluid used for BAL was not described in the study; it is possible that a difference in lavage fluid volume affected the result of each test. Standardization of the method for bronchial washing is desirable; however, it might be difficult, as the amount of fluid recovered varies depending on the location of the focus of infection in the lungs. Due to the nature of the lateral-flow device’s qualitative test, there could be a difference in interpreting the positive line.
This study has several limitations, including the small size of the patient cohort and the fact that it is a single-center retrospective study. Another limitation is that Aspergillus IgG antibody ELISAs were not used for comparison, as these are not commercially available in Japan. The essential limitation in this research area is the difficulty of CPA diagnosis, as gold standards of diagnosis, such as histopathological findings and fungal culture tests, are difficult to satisfy; additionally, the utility of the Aspergillus antibody test for non-fumigatus Aspergillus spp. might be limited.
In conclusion, to the best of our knowledge, this is the first study to evaluate the performance of the AspLFD in serum for the diagnosis of CPA. Given the results reported here, the performance of AspLFD was not sufficiently good but was tolerable for the diagnosis of CPA; moreover, the best advantage of this test over other serodiagnostic tests is its short turnaround time, the most important requirement for point-of-care test. The AspLFD has potential as a screening test for CPA in clinics or resource-limited countries, but further prospective studies with large numbers of patients are required.
ACKNOWLEDGMENTS
Newly improved LFD tests used in this study were purchased from OLM Diagnostics in 2018. OLM Diagnostics was not involved in planning the study design, in data collection, analysis, or interpretation, or in the writing of the manuscript.
T. Takazono and K. Izumikawa planned the research design and drafted the manuscript. Y. Ito and K. Nishimura tested the patients’ samples. M. Tashiro, T. Saijo, K. Yamamoto, Y. Imamura, and T. Miyazaki collected patient’s clinical data. K. Yanagihara and H. Mukae revised the manuscript. All authors read and approved the submitted version.
The authors declare no conflicts of interest associated with the manuscript.
FOOTNOTES
- Received 19 January 2019.
- Returned for modification 11 February 2019.
- Accepted 27 February 2019.
- Accepted manuscript posted online 6 March 2019.
- Copyright © 2019 American Society for Microbiology.
