Patients and Methods

Patients
Adult patients (n = 29) followed in our French cystic fibrosis care center and the lung diseases department (Assistance Publique—Hôpitaux de Marseille) were routinely assessed for ABPM diagnosis between April 2017 and January 2018. Patients were categorized in accordance with the final diagnosis as ABPA, Af-sensitized (AF-S), fungal colonization, or control CF patients. These categories were defined as follows: ABPA met all the ISHAM criteria (4); AF-S displayed sIgE to Af (0.1 kUA/L or greater) without fulfilling the ISHAM criteria for ABPA; fungal colonization was defined as at least one filamentous fungus cultured from a bronchial sample during the previous 6 months, without fulfilling the ISHAM criteria, while patients who were categorized in none of the previous categories were considered as control CF patients. Demographic and laboratory data for the study cohort are detailed in Table 1.
Table 1  Demographic and laboratory findings of the study cohort.
Control   Af-sensitized   ABPA  Fungal colonization   Total
n  16  3  3  7  29
Age (years) (median ± 5–95 percentile)  43 (26-61)  35 (27-44)  21 (17-24)  42 (22-62)  38 (18-62)
Male/female  7/9  1/2  0/3  5/2  13/16
Lung transplantation  12/16  3/3  0/3  4/7  19/29
Time since transplantation (median ± 5–95 percentile)  6 (1-19)  7 (4-14)  /  2 (1-10)  6 (1-17)
Bacterial colonization  9/16  1/3  3/3  6/7  19/29
Fungal colonization  7/16  2/3  3/3  6/7  18/29
Chest HRCT abnormality(ies)  14/16  2/3  3/3  7/7  26/29
Total IgE (kIU/L) (median ± 5–95 percentile)  73 (4–333)  72 (41–100)  414 (74–854)  12 (2–26)  94 (2–469)
IgE Af (kUA/L) (median ± 5–95 percentile)  <0.1  1 (0.1–2)  10 (3–19)  <0.1  1 (0–6)
Eosinophils (mm3) (median ± 5–95 percentile)  321 (107–623)  327 (161–602)  370 (135–775)  319 (149–565)  326 (128–692)
ABPA, Allergic bronchopulmonary aspergillosis; Af, Aspergillus fumigatus; HRCT, High-resolution computed tomography.

Functional Cytometric Tests
All the assays were done with Af, Penicillium notatum (Pen), and Alternaria alternata (Alt) extracts (Bühlmann Laboratories®, Schönenbuch, Switzerland). BAT was performed with the Flow2CAST method (Bühlmann Laboratories®), using CCR3 (CD193) and CD63 as basophil identification and activation markers, following the manufacturer's instructions. Positive controls were anti-RFcεI and the bacterial peptide fMLP. For LST, whole blood was incubated in a 96-well plate with RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) and sequential allergen dilution for 24 h under 5% CO2. Phytohemagglutinin (PHA, Thermo Fisher Scientific), 10 μg/L, was used as a positive control. Each well was harvested and stained with a mix of the following antibodies: PerCP-anti-CD45 (clone 2D1), FITC-anti-CD3 (clone SK7), APC-anti-CD4 (clone SK3), PE-anti-CD8 (clone SK1), and PeCy7-anti-CD69 (clone L78) (BD Biosciences®, San Diego, California). Flow cytometry was performed on a FACS Canto II (Becton Dickinson, Le Pont de Claix, France) and at least 200 basophils per sample were analyzed for BAT and 10,000 lymphocytes for LST.

Data Expression
Data were analyzed using FACS Diva software (TreeStar, Ashland, OR). Specific IgE (sIgE) to Af extract levels were measured with the Thermo Fisher ImmunoCAP platform (Phadia, Thermo Fisher Scientific, Uppsala, Sweden). All the results were expressed as the basophil or lymphocyte stimulation index, which is the ratio between level of activation with the allergen and level of activation with reaction buffer, with a threshold of 2. Statistical analysis was performed with the R statistical software (14). A correlation matrix was calculated using Pearson's correlation. Mean responses of each group were compared via the Student or Kruskal–Wallis test, a two-sided p < 0.05 was statistically significant.

Ethics Statement
The study was based on a retrospective review of medical charts and laboratory results. Under the French law, ethics committee approval and patient consent were not required for this type of non-interventional study, provided the patients had received information and retained the right to oppose the use of anonymized medical data (15, 16).