We collected sputum samples and cough plates from 15 cystic fibrosis patients in the Netherlands who were colonized with Aspergillus fumigatus; we recovered A. fumigatus of the same genotype in cough aerosols and sputum samples from 2 patients. The belief that transmission of A. fumigatus from cystic fibrosis patients does not occur should be reconsidered.

Aspergillus fumigatusis a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical forA. fumigatusto adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply inA. fumigatusdue to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis ofA. fumigatusisolates cultured from different origins.As diploids are the hallmark of parasex, we screened 799A. fumigatusisolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation.We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acuteAspergillusinfection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate.Parasexual recombination allowsA. fumigatusto adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology ofA. fumigatusin the human lung.

Cystic fibrosis (CF) can be complicated by fungal infection of the respiratory tract. Fungal detection rates in CF sputa are highly dependent on the culture protocol and incubation conditions and thus may lead to an underestimation of the true prevalence of fungal colonization. We conducted a prospective study to evaluate the additional value of mucolytic pre-treatment, increased inoculum (100 μL), additional fungal culture media (Sabouraud agar; SAB, Medium B+, Scedosporium selective agar; SceSel+ and Dichloran-Glycerol agar; DG18) and longer incubation time (3 weeks) compared with our current protocol. Using the new protocol, we prospectively analyzed 216 expectorated sputum samples from adult and pediatric CF patients (n = 77) and compared the culture yield to a three year retrospective cohort that used direct 10 μL loop inoculation on SAB with 5 days incubation (867 sputum samples/103 patients). Detection rates for molds increased from 42% to 76% (p < 0.0001). Twenty-six percent of cultures were polymicrobial in the prospective cohort as opposed to 4.7% in the retrospective cohort (p < 0.0001). Colonization rate with A. fumigatus increased from 36% to 57%. SAB and DG18 showed the highest detection rates for all molds (SAB 58.6%; DG18 56.9%) and DG18 had the best performance for molds other than A. fumigatus. The larger sample volume and longer incubation also contributed to the increased recovery of molds. The introduction of a modified fungal culture protocol leads to a major increase in detection rate and the diversity of molds, which influences fungal epidemiology and may have implications for treatment decisions.

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR₃₄/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR₃₄³/L98H evolved from a clinical TR₃₄/L98H ancestor isolate, confirmed by whole genome sequencing. TR₃₄³/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR₃₄³, thus supporting the possibility of in-host selection of TR₃₄ variants.

Penicillium and Talaromyces species are frequently isolated from the respiratory tracts of cystic fibrosis (CF) patients, yet their diversity, ecological roles, and clinical significance remain poorly understood. In this study, we analyzed 521 fungal isolates (482 Penicillium and 39 Talaromyces ) obtained from Dutch CF patients to investigate species diversity and prevalence. Using a combination of AFLP fingerprinting and DNA sequences analysis, we identified 57 Penicillium and 18 Talaromyces species, including a putatively new species named Penicillium subluteum sp. nov. The most commonly isolated Penicillium species included P. crustosum , P. frequentans , P. chrysogenum , P. rubens , and P. brevicompactum , while Talaromyces rugulosus was the most prevalent Talaromyces species. Our findings highlight the underestimated diversity of Penicillium and Talaromyces in CF patients and emphasize the importance of accurate species identification for understanding fungal colonization patterns and assessing pathogenic potential. This study provides the most comprehensive overview to date of Penicillium and Talaromyces diversity in the CF airway and contributes valuable taxonomic and ecological insights into the role of these fungi in patients with chronic airway disease.