In vivo molecular microimaging of pulmonary aspergillosis.

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.

In vivo and in situ imaging of experimental invasive pulmonary aspergillosis using fibered confocal fluorescence microscopy.

Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunosuppressed patients. In this study, we assessed fibered confocal fluorescence microscopy (FCFM), a new endoscopic technique that enables in vivo microscopic imaging of the distal lung, as a tool for in vivo imaging of IPA. IPA was induced in immunosuppressed rats using a wild strain of Aspergillus fumigatus (n = 6) or a fluorescent transformed TAG-RFP A. fumigatus strain (n = 10). Subpleural areas of pulmonary infection were imaged in vivo using FCFM employing a transthoracic approach. Results were compared to three immunosuppressed control groups, i.e., non-inoculated rats (n = 4), rats inoculated with sterile Phospate-buffer saline (PBS; n = 5), and rats inoculated with Geosmithia argillacea (n = 6). Only hyphae of TAG-RFP A. fumigatus were detectable both in vitro and in vivo by FCFM. In vivo, a local infiltration of fluorescent alveolar macrophages was observed with FCFM in IPA areas in all fungal infections groups, but also in focal inflammatory areas in the immunosuppressed PBS group. A specific fibrillar fluorescence was observed in IPA areas with the TAG-RFP A. fumigatus group, with a 83% sensitivity, a 100% specificity, a 100% positive predictive value and 94% negative predictive value. FCFM provides a new tool to study host-aspergillus interactions in vivo.