Patients and data collection
Fifty-eight 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 the diagnosis and treatment of pulmonary mycosis and the criteria of the European Organization for Research and Treatment of Cancer (EORTC) [11, 12]. The exclusion criteria were as follows: (1) agranulocytic patients, (2) patients with other lung diseases, (3) patients with possible pulmonary aspergillosis, (4) patients with allergic bronchopulmonary aspergillosis, and (5) patients who were positive for human immunodeficiency virus (HIV).
The diagnostic criteria of IPA included the following: (1) patients had risk factors for pulmonary aspergillosis (such as neutropenia, transplantation, and immunosuppressive therapy), (2) patients had certain clinical manifestations of IPA, (3) imaging results were abnormal, and (4) there was microbiological evidence of IPA.
The diagnostic criteria of CPA were as follows: (1) chronic lung symptoms (cough, expectoration, haemoptysis, 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 of CPA (the culture of sputum, bronchoalveolar lavage fluid and bronchoscopy was positive and the blood G test and GM test were positive), and (4) no or a 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 the control groups were not significantly different from those of the pulmonary aspergillosis group. The following data were collected: demographic data (age, sex, weight), serum indexes, imaging features, biochemical indicators, bacterial and fungal culture results, bronchoscopic findings, and treatment outcomes. In addition, all participants signed informed consent voluntarily, and the study was approved by the ethics committee of Tianjin Chest Hospital (protocol number: 2018KY-009-01).
Five millilitres of venous blood was extracted before the administration of any antibiotics. Serum was separated from the blood for immediate testing or was stored frozen at −80°C for later testing.
The serum (1,3)-β-D-glucan test (G test) was conducted with a chromogenic method using a (1-3)-β-D-glucan detection kit (Dynamiker Biotechnology Co., Ltd, Tianjin, China) . In brief, a 5 μl serum sample was first pretreated for 10 min at 37°C with 20 μl of a solution containing 0.6 M KCl and 0.125 M KOH and then assayed with 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.
The serum galactomannan test (GM test) was carried out with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Dynamiker Biotechnology Co., Ltd. Tianjin, China) according to the manufacturer's instructions. The judgement criteria for the GM test results were as follows: ≥ 0.85 µg/L was considered positive, < 0.65 µg/L was considered negative, and 0.65-0.85 µg/L was considered intermediate.
The commercial ELISA kit (Dynamiker Biotechnology Co., Ltd. Tianjin, China) was used to detect Aspergillus IgG antibody, and the experimental procedure followed the instructions. An Aspergillus IgG concentration ≥ 120 AU/ml was considered positive, < 80 AU/ml was considered negative, and 80-120 AU/ml was considered intermediate.
According to the manufacturer's instructions, Aspergillus IgM antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Dynamiker Biotechnology Co., Ltd. Tianjin, China). The judgement criteria for Aspergillus IgM detection included the following: ≥ 120 AU/ml was considered positive, < 80 AU/ml was considered negative, and 80-120 AU/ml was considered the intermediate.
SPSS 21.0 software was used for statistical analysis. Comparisons between groups were performed by the chi-squared test. Fisher's test results were used when the sample size was small and the theoretical number was small. The Mann-Whitney U test was used in the course of disease, age and serum indicators except lymphocyte count indicators. An independent-sample t test was used for lymphocyte count indicators. The sensitivity, specificity and optimal threshold were determined by receiver operating characteristic (ROC) curve analysis in the pROC package. The best cut-off value was the value that maximized the sum of the sensitivity and specificity in the ROC curve. This study defined a P value <0.05 as a significant difference.