Preliminary Identification of Clinical <i>Aspergillus flavus</i> Isolates Using a Rapid β-Tubulin PCR–RFLP Method and Evaluation of Virulence Factors

Abstract

Background: Among Aspergillus species, A. flavus is frequently associated with invasive and noninvasive aspergillosis, and it produces several virulence factors, including extracellular enzymes. Objectives: This study evaluated the utility of β-tubulin PCR–restriction fragment length polymorphism (PCR–RFLP) with the restriction enzyme AlwI for the preliminary identification of clinical A. flavus isolates and examined selected virulence factors. Methods: Forty-five archived clinical isolates initially identified as A. flavus were retrieved from bronchoalveolar lavage (BAL), sinus discharge, and nail specimens. The β-tubulin gene was amplified by PCR, and the resulting amplicons were digested with AlwI; representative isolates were confirmed by sequencing. Protease and phospholipase activities were assessed on appropriate media, and enzyme activity indices (EAI) were calculated. Group differences were analyzed using the Kruskal–Wallis test, followed by Dunn’s pairwise test with Bonferroni adjustment. Results: PCR–RFLP produced identical two-band patterns (296 bp and 254 bp) in all isolates, and sequencing confirmed species identification in the selected subset. A phospholipase-positive phenotype was observed in 57.8% of isolates, with no statistically significant variation by specimen source (H(2) = 4.965, P = 0.084). Proteolytic activity was positive in 71.1% of isolates and showed significant dependence on specimen source (H(2) = 6.909, P = 0.032, η2H = 0.117); Dunn’s pairwise test with Bonferroni adjustment demonstrated higher protease activity in sinus isolates than in BAL isolates (adjusted P = 0.029). Conclusions: β-tubulin PCR–RFLP using AlwI is a rapid and practical preliminary method for identifying A. flavus; however, sequencing remains necessary for definitive species identification. Variation in extracellular enzyme production among anatomical sources suggests phenotypic heterogeneity relevant to pathogenic potential.