Role of Real time PCR and Galactomannan in the classification of Aspergillus disease in CF

Purpose Cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) are routinely treated with azole antifungals. However, it is not known whether Aspergillus colonised or sensitised patients would similarly benefit from antifungal treatment. To aid these treatment decisions and monitor treatment response, accurate methods to detect Aspergillus in sputum are first needed. This study aimed to validate and then integrate real time PCR and galactomannan (GM) antigen detection, from CF sputum, with serological analysis to identify patients groups that may benefit from antifungal therapy. Methods 146 adult CF patients provided a sputum sample and a blood sample. 30 of these patients provided a second sputum sample within 6 months. Serological tests included total IgE (tIgE), specific A.fumigatus IgE (sIgE) and specific A.fumigatus IgG (sIgG) performed by Phadia ImmunoCAP® assay. Sputum samples were homogenised with sputasol (Oxoid, UK) and sonication for 120 seconds (Sonics® VC505). 10µL was cultured on sabouraud agar with chloramphenicol (Oxoid, UK) for 72 hours at 37ºC. 300µL was used in the Platelia™ Aspergillus EIA kit (Bio-Rad, Marrnes-La-Cocquette, France) to measure GM antigen, the cut-off optical index of ≥ 0.5 being used to define positivity. The remaining sputum sample had fungal DNA extracted using the MycXtra™ DNA extraction kit (Myconostica, Manchester, UK) and then Aspergillus DNA was detected using the real time PCR kit MycAssay™ Aspergillus (Myconsotica, UK). Latent class analysis was used to define patient groups using Mplus version 6.1 software.Results 37% (39) of the 146 sputum samples were positive for Aspergillus species by standard culture whereas 74% (108) were positive for Aspergillus species using real time PCR. Repeatability over 6 months was performed in 30 patients. All PCR positive patients remained positive and 5 negative patients became positive. PCR repeatability within a sputum sample was performed for 10 samples. Repeatability was 100% with Ct values varying by