Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry

Abstract

Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

FIG 1

Cultures and microscopic examinations of clinical isolates on SDA after 5 days at 37°C. PW2952 (A) and PW2955 (B) produced velvety colonies with floccose tufts and yellowish green conidia. (C) PW2958 produced dark green-brown conidia at maturity and formed velutinous colonies with floccose tufts. The conidiophores of PW2952 (D) and PW2955 (E) were hyaline, with globose to subglobose vesicles and biseriate phialides. (F) The conidiophores of PW2958 were hyaline, with globose vesicles and biseriate phialides producing brownish-yellow conidia.

FIG 2

Phylogenetic analysis of ITS (336 nucleotide positions), β-tubulin gene (219 nucleotide positions), and calmodulin gene (199 nucleotide positions) of the 11 clinical isolates, three reference strains, and other closely related species. Trees were constructed by the maximum likelihood method with 1,000 bootstrap replicates. Only bootstrap values of ≥60% are shown. Scale bars indicate the estimated number of nucleotide substitutions per 100, 20, and 20 nucleotides, respectively.

FIG 3

(A) Principal component analysis and (B) hierarchical clustering dendrogram of metabolic fingerprints of the 11 clinical isolates reported as A. flavus and three reference strains. Molecular features (1,016) defined by retention time and mass pair were included. The 3D PCA score plot for component PC1 versus component PC2 versus component PC3 is presented. The percentages of variance in the data set reflected by the first three PCs for each sample are shown. Hierarchical clustering of different fungal isolates is represented by a heat map and a dendrogram. Clustering was performed using Euclidean distance and average linkage. Blue and red indicate negative and positive log ratios, respectively. Two biological replicates were performed for each sample. Lanes 1 and 2, PW2952; lanes 3 and 4, PW2956; lanes 5 and 6, PW2957; lanes 7 and 8, PW2960; lanes 9 and 10, PW2962; lanes 11 and 12, ATCC 204304; lanes 13 and 14, PW2953; lanes 15 and 16, PW2954; lanes 17 and 18, PW2961; lanes 19 and 20, CBS260.88^T; lanes 21 and 22, PW2955; lanes 23 and 24, PW2959; lanes 25 and 26, CBS104.13^T; lanes 27 and 28, PW2958.

FIG 4

Hierarchical clustering of MALDI-TOF MS spectra of 11 clinical isolates reported as A. flavus and three reference strains. Distances are displayed in relative units.