Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

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. fumigatussensu 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. fumigatussensu 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.

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples

“…Notwithstanding, as stated above, the currently available approaches to routine do not fully comply with the requirements for Legionella testing. FISH can take a step forward as a powerful and rapid tool for identification of microbial populations without the need for time-consuming microorganisms culturing [ 13 , 40 , 41 ]. Additionally, PNA-FISH overcomes problems related to PCR-based molecular technologies, such as susceptibility to inhibitors, cross-contamination, false positive results and even the requirements for specialized personnel [ 24 , 42 ].…”

“…Hybridization was performed as previously described by the group [ 13 , 35 , 36 ], with some modifications. As Figure 2 A summarizes, suspensions of 2 × 10 7 cell/mL of L. pneumophila serogroup 1 (WDCM 00107) and L. anisa ATCC 35,292 were dispensed in 8 mm well slides (Marienfeld, Lauda-Königshofen, Germany) and allowed to air dry.…”

“…These molecular methods can detect the number of genome units (GU) per liter, but there is still a need for correspondence with the number of colony forming units (CFU), which is usually lower, probably due to VBNC cells’ presence [ 11 ]. Research has been conducted in order to develop, optimize and validate new molecular methods, and fluorescence in situ hybridization (FISH) has been shown to be a promising technology [ 12 , 13 , 14 , 15 , 16 ].…”

“…Moreover, they present a stronger affinity to the target sequences and subsequently higher accuracy, mainly explained by the fact that these molecules are neutrally charged, reducing the electrostatic repulsion that occurs between negatively charged DNA/DNA or RNA/RNA duplexes [ 21 , 22 ]. PNA-FISH has been used for the rapid detection (approximately 3 h) of several relevant microorganisms, such as bacteria [ 23 , 24 , 25 , 26 , 27 ], yeasts [ 28 , 29 ] and filamentous fungi [ 13 , 30 , 31 ]. Regarding Legionella , only one Legionella spp.…”

Aspergillus and Health

“…The authors reported that, using Flamingo Medium, they were able to recover an average of 20−30% more A. fumigatus colonies compared to other media, and at the same time, a 95% reduction in the number of Mucorales colonies was also observed. Also with the purpose of improving the diagnosis of aspergilosis, Cerqueira et al [ 2 ] presented a method developed for detection of A. fumigatus sensu stricto in respiratory samples using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH). The sensitivity of the tested probe was 100% in strains but 79% in clinical samples, with a specificity of 100% in both cases.…”