Abstract
Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatus sensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatus sensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.
Highlights
Aspergillus fumigatus is a saprophyte filamentous fungus that feeds on decaying organic matter and is able to form a type of spore, named conidium, which can survive in a wide range of aggressive environments and spread through the air [1,2]
An accurate early diagnosis of A. fumigatus in clinical samples such as bronchoalveolar lavage fluid (BAL), sputum and blood, among others, is crucial for a more rational and successful treatment of Invasive Aspergillosis (IA) [8,9]. Diagnosis of this microorganism relies on non-specific techniques, such as direct microscopy visualization and serologic tests based on enzyme-linked immunosorbent assay (ELISA) (that target the fungi cell wall components galactomannan and (1,3)-β-D glucan), or on fastidious and time-consuming culture methods [9,10]
Strains were provided by Colección Española de Cultivos Tipo (CECT), Micoteca da Universidade do Minho (MUM), and the American Type Culture Collection (ATCC)
Summary
Aspergillus fumigatus is a saprophyte filamentous fungus that feeds on decaying organic matter and is able to form a type of spore, named conidium, which can survive in a wide range of aggressive environments and spread through the air [1,2]. Diagnosis of this microorganism relies on non-specific techniques, such as direct microscopy visualization and serologic tests based on enzyme-linked immunosorbent assay (ELISA) (that target the fungi cell wall components galactomannan and (1,3)-β-D glucan), or on fastidious and time-consuming culture methods [9,10]. PCR-based molecular techniques have been applied in A. fumigatus detection, but a lack of methodological standardization, the occurrence of false positive results and the discrepant results due to the Ct interpretation values are hindering a more widespread use of this technique [9,11]. Fluorescence in situ hybridization (FISH) is another molecular-based method that can be applied directly in clinical samples [12], allowing the visual identification of microorganisms and the detection of viable cells with greater certainty, which may decrease the number of false positives
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