The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals

Abstract

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the β-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial β-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

Keywords: Aspergillus flavus; Aspergillus nomiae; Aspergillus parasiticus; Aspergillus sojae; Flavi section; antifungal resistance; cryptic species.

Figure 1

Phylogenetic tree of A. flavus of concatenated partial β-tubulin and calmodulin sequences. (A) All isolates and reference sequences. (B) Only A. flavus ss with A. aflatoxiformans as outgroup. The evolutionary history was inferred using the Neighbor-joining method [61]. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [62]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [63] and are in the units of the number of base substitutions per site. This analysis involved 175 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 970 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 [64].