Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study

Abstract

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: "MucoFong" program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran-rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37 °C and on Sabouraud-chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20-27 °C. Each plate was checked twice a week during 3 weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16 days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

The myriad challenges of respiratory fungal infection in cystic fibrosis

“…of 40.8%, Scedosporium of 5.2%, Exophiala 4.7%, and Trichosporon 3.3% . Figure demonstrates that the prevalence of filamentous fungi isolated using fungal selective media is similar across these recent studies …”

“…While a shorter period of incubation may be sufficient for some fungal species, emerging organisms may require much longer culture duration; ultimately the authors of the MucoFong study recommended an incubation time of 16 days . However, other authors have concluded 7 days may be long enough …”

Fungal Respiratory Infections in Cystic Fibrosis (CF): Recent Progress and Future Research Agenda

“…Similarly, panfungal PCR, DNA sequencing, MLST and mass spectrometry methods are reviewed as the solid foundations for the diagnosis of fungal disease in CF [12]. Delhaes and the group from the ''MucoFong'' program summarize the results of systematic investigation on the optimal culture methods for recovery of fungal pathogens from the respiratory specimens of CF patients [13]. This [5,6] 2.…”

“…This [5,6] 2. Fungal diagnostic criteria in CF patients [6,9,12,13] 3. Antifungal therapy in CF patients [7,9,[14][15][16][17] 4.…”

“…The investigators found that about half of the patients presented with at least one fungal species, while 37% were co-colonized with two species. The investigative team recommended a standardized protocol with the use of four culture media to facilitate the growth of Aspergillus species and yeasts, and slow-growing molds with a 16-day incubation period [13]. Hector et al [14] also write critically about the unsolved issues around fungal colonization, sensitization or invasion of CF airways.…”