Detection of Aspergillus fumigatus hyphae in respiratory secretions by membrane filtration, fluorescent labelling and laser scanning

Abstract

The detection of Aspergillus fumigatus hyphae in bronchoalveolar lavage fluid (BAL) and sputum is diagnostically useful in patients at risk of invasive aspergillosis. We report a dedicated enzymatic-chemical sample pretreatment that allows the application of a previously described solid phase cytometry (SPC) method for detection of A. fumigatus hyphae in sputum and BAL samples. Non-specific detection of fungal hyphae by SPC is based on a ‘viability’ staining using carboxyfluorescein diacetate. For a specific detection of A. fumigatus hyphae by SPC, viability staining is combined with a pre-incubation at 45 °C, immunofluorescent labelling and microscopic recognition of the characteristic hyphal morphology. Low numbers of A. fumigatus hyphae (2–10 hyphae/sample) have now been demonstrated in spiked sputum using the non-specific and specific staining in 2.5 and 8.5 h, respectively.