Patients and data collection
58 pulmonary aspergillosis cases in non-agranulocytic patients admitted to Tianjin Chest Hospital from July 2017 to July 2018 were enrolled. The diagnostic criteria referred to the consensus of experts in diagnosis and treatment of pulmonary mycosis and the criteria of the European Organization for Research and Treatment of Cancer (EORTC) [10, 11]. The exclusion criteria were as follows: (1) other lung diseases; (2) patients with possible pulmonary aspergillosis; (3) patients of allergic bronchopulmonary aspergillosis; (4) human immunodeficiency virus (HIV) was positive.
The diagnostic criteria of IPA included: (1) patients had risk factors for pulmonary aspergillosis (such as neutropenia, transplantation, and immunosuppressive therapy, etc.); (2) patients had certain clinical manifestations; (3) imaging is abnormal; (4) microbiology evidence.
The diagnostic criteria of CPA were as follows: (1) chronic lung symptoms (cough, expectoration, hemoptysis, weight loss) for more than 3 months; (2) progressive imaging abnormalities (new or progressive cavities, infiltration around the cavity, thickening of the pleura, fungal balls); (3) microbiological evidence (the culture of sputum, bronchoalveolar lavage fluid and bronchoscopy showed positive, blood G test and GM test were positive); (4) no or low degree of immune impairment.
During the same period, 15 cases of community-acquired bacterial pneumonia and 50 healthy individuals served as control groups. The sex and age of control groups had no significant difference compared with pulmonary aspergillosis group. Moreover, the following data were collected: demographic data (age, gender, weight), serum indexes, imaging features, biochemical indicators, bacterial and fungal culture results, bronchoscopic findings, and the treatment outcomes. In addition, all participants has signed the informed consent voluntarily, and the study has been approved by the ethics committee of Tianjin Chest Hospital (protocol number: 2018KY–009–01).
Serological testing5 mL venous blood was extracted before using any antibiotics. Serum was separated from the blood for tests directly or stored frozen at −80°C.
G test
The serum (1,3)-β-D-glucan test (G test) was conducted with a chromogenic method using (1–3)-β-D-glucan detection kit (Dynamiker Biotechnology Co., Ltd, Tianjin, China) [12]. In brief, 5 μl serum sample was firstly pretreated for 10 min at 37°C with 20 μl of a solution containing 0.6 M KCl and 0.125 M KOH, and assayed with the Glucatell reagent in a kinetic, chromogenic format for 30 min at 37°C. Subsequently, the optical densities at 405 nm (OD405) were read. Finally, the concentration of G in each sample was calculated by using a calibration curve with standard solutions of 6.25 to 100 pg/ml. Cases were judged positive if the level of G was ≥120 pg/ml in at least one serum sample.
GM test
The serum galactomannan test (GM test) was carried out with commercial enzyme-linked immunosorbent assay (ELISA) kit (Dynamiker Biotechnology Co., Ltd. Tianjin, China) according to the manufacturer’s instructions. The judgment criteria of GM test results were as follows: ≥ 0.85 ug/L was positive, < 0.65 ug/L was negative, and 0.65–0.85 ug/L was intermediate.
Aspergillus IgG
The commercial ELISA kit (Dynamiker Biotechnology Co., Ltd. Tianjin, China) was used to detect Aspergillus IgG antibody, and the experimental procedure was relied on the instructions. Aspergillus IgG concentration ≥ 120 AU / ml was positive, < 80 AU / ml was negative, 80–120 AU / ml was the intermediate.
Aspergillus IgM
According to the manufacturer’s instructions, Aspergillus IgM antibody was detected by the commercial enzyme-linked immunosorbent assay (ELISA) kit (Dynamiker Biotechnology Co., Ltd. Tianjin, China). The judgment criteria of of Aspergillus IgM included: ≥ 120 AU / ml was positive, < 80 AU / ml was negative, 80–120 AU / ml was the intermediate.
Statistical analysis
SPSS 21.0 software was used for statistical analysis. Comparison between groups were performed by chi-square test. Fisher’s test results were used when the sample size was small and the theoretical number was small. Mann-Whitney U test was used in the course of disease, age and serum indicators except lymphocyte count indicators. Independent sample t test was used for lymphocyte count indicators. The sensitivity, specificity and optimal threshold were determined by using the receiver operating characteristic curve (ROC curve). The standard of this study was P < 0.05 with statistical difference.