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

Abstract

BackgroundWithin 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.MethodsNinety-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.ResultsThe 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).ConclusionsThe 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.

Simple, Low-Cost Molecular Assays for TR 34 /L98H Mutations in the cyp51A Gene for Rapid Detection of Triazole-Resistant Aspergillus fumigatus Isolates

“…PCR cycling (total, 35 cycles) included denaturation at 95°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 1 min. An initial denaturation step at 95°C for 5 min and a final extension step at 72°C for 10 min were also included, and the amplicons were detected by use of 2% agarose gels (16). A. fumigatus isolates containing TR 34 in the cyp51A promoter region should yield an amplicon of 139 bp, while isolates containing the wildtype sequence (no tandem repeat) should yield an amplicon of 105 bp.…”

“…The details of clinical/environmental A. fumigatus isolates from France, The Netherlands, and India that were used in this study have also been published previously (4,14,15). Drug susceptibility testing (DST) of A. fumigatus isolates with itraconazole was carried out by Etest as described elsewhere (16). Isolates with reduced susceptibility to itraconazole (MIC of Ն2 g/ml) were also tested for voriconazole by a broth microdilution (M38-A2) method (4).…”

“…PCR amplicons from all A. fumigatus isolates were also sequenced to confirm the results. Sequencing reactions were carried out and data were analyzed as described previously (16). In agarose gels, PCR amplification of the promoter region yielded distinct 105-bp and 139-bp amplicons from reference A. fumigatus isolates CBS 113.26 (containing the wild-type sequence) and VPCI1042/09 (containing TR 34 in the promoter region), respectively.…”

Use of Novel Tools to Probe Drug Resistance in Fungi

“…In principle, a primary sample like blood or tissue specimen can be analyzed in a matter of hours to generate species-specific information, which predominantly involves amplification-detection platforms, including polymerase chain reaction (PCR) (DNA amplification) and nucleic acid sequence-based amplification (RNA amplification)-based assays. Various molecular tools, in particular, postamplification reporting methods like allele-specific molecular beacon (MB) technology, DNA sequencing, and melt curve analysis have been incorporated with these amplification techniques for species identification (Al-Wathiqi et al 2013;Loeffler et al 2000a;Park et al 2000). Of note, some new high-throughput technologies seem more attractive, such as Luminex xMAP technology, which utilizes microbeads and specific capture probe hybridization to identify up to 100 different target sequences in a single reaction vessel (Loeffler et al 2000b).…”

Aerial Prevalence of Aspergillus calidoustus Isolates in and around a Tertiary Care Hospital in Kuwait and Assessment of Their Pathogenicity

“…Genomic DNA from the isolates was prepared as described previously and used as the template for PCR amplification (6). The ITS (ITS-1, 5.8 S rRNA, and ITS-2) regions of rRNA genes and variable regions of ␤-tubulin and calmodulin gene fragments were amplified and sequenced as described previously (7,8). GenBank Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches were performed for species identification.…”