Molecular identification and antifungal susceptibility profile of Aspergillus flavus isolates recovered from clinical specimens in Kuwait.

Background: Within the genus Aspergillus, A. flavus is the second most important species of clinical significance. It is predominantly associated with infections involving sinuses, eye and skin, mostly in geographic regions with hot and arid climate, including the Middle East. Recent reports on emergence of resistance to triazoles among Aspergillus spp. is a cause of concern for treatment of patients with invasive aspergillosis. In this study we present data on genetic characterization and antifungal susceptibility profile of clinical and environmental isolates of A. flavus.

Methods: Ninety-nine Aspergillus section Flavi isolates, originating from clinical (n=92) and environmental (n=7) sources, initially identified by morphological characteristics, were analyzed by partial sequencing of β-tubulin and calmodulin gene fragments and their susceptibilities to six antifungal agents was determined by Etest on RPMI1640 and Muller-Hinton agar media. Etest minimum inhibitory concentrations (MICs) of amphotericin B and voriconazole were also compared with zone of inhibition diameters obtained by disc diffusion test on RPMI agar medium.

Results: The identity of all clinical and environmental isolates was confirmed as A. flavus species by combined analysis of β-tubulin and calmodulin genes. The mean MIC90 (μg/ml) values on RPMI medium for amphotericin B, voriconazole, posaconazole, anidulafungin, micafungin and caspofungin were 3, 0.25, 0.25, 0.002, 0.002 and 0.032, respectively. No environmental isolate exhibited MIC value of >2 μg/ml for amphotericin B. For clinical isolates, the zone of inhibition diameters for amphotericin B and voriconazole ranged from 7-16 mm and 24-34 mm, respectively. Linear regression analysis between Etest MIC values and disk diffusion diameters revealed a significant inverse correlation with amphotericin B (p <0.001) and voriconazole (p<0.003).

Conclusions: The β-tubulin and calmodulin gene sequences confirmed that all 92 clinical isolates identified phenotypically belonged to A. flavus taxon, thus suggesting that the other species within Aspergillus section Flavi are of little clinical significance. Triazoles and echinocandins showed very good in vitro activity against the A. flavus, however, 10% clinical isolates showed MICs of >2 μg/ml for amphotericin B.