Development of an Oligonucleotide Array for Direct Detection of Fungi in Sputum Samples from Patients with Cystic Fibrosis

Abstract

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.

Aspergillus Species and Other Molds in Respiratory Samples from Patients with Cystic Fibrosis: a Laboratory-Based Study with Focus on Aspergillus fumigatus Azole Resistance

“…Other centers have reported that S. apiospermum and A. terreus are the second and third most frequently observed filamentous fungi (42). However, the routine media and incubation time used in our study are not optimal for species like S. apiospermum and E. dermatitidis, and thus, the prevalence rates of these are probably underestimated (9,38).…”

Phenazine-1-Carboxylic Acid Promotes Bacterial Biofilm Development via Ferrous Iron Acquisition

“…The full complexity of the microbial community in the CF lung and the interactions among its members are only beginning to be appreciated (8,25,63). Systematic studies are needed to understand how iron availability-impacted by both host cells and the microbiome-affects this ecosystem.…”

Purification and Characterization of a Mycelial Catalase from Scedosporium boydii, a Useful Tool for Specific Antibody Detection in Patients with Cystic Fibrosis

“…As for mycological examination, it requires skill and expertise and may lead to false-negative results for polymicrobial specimens like sputum samples because of the more rapid and more extensive growth of other molds frequently associated with this fungus, like Aspergillus fumigatus (19). Several molecular methods have been proposed for detection of the fungus from sputum samples (20)(21)(22)(23)(24), but as culture methods, they do not allow the differentiation between airway colonization and sensitization of the patient or respiratory infection in the CF context, which has important implications for patient management.…”