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Title: Phenotypic and Molecular Characterization of Aspergillus Section Flavi Isolates Recovered from Clinical Specimens in Kuwait
Authors: Faten Ali Al-Wathiqi 
Supervisor: Prof. Ziauddin Khan
Keywords: phenotypic characterization;Molecular characterization;Aspergillus section Flavi;clinical specimens;Kuwait
Issue Date: 2012
Publisher:  Kuwait university - college of graduate studies
Abstract: Abstract More than 200 species (including ~20 human pathogens) of Genus Aspergillus, ubiquitous molds widely distributed in the environment, have been described in the literature. Majority of invasive infections are caused by Aspergillus fumigatus, however, isolates belonging to Aspergillus section Flavi are second most frequent pathogenic species. Aspergillus flavus is one of seven and an important clade of Aspergillus section Flavi as these species are quite prevalent in dry and arid climate of the Middle East. The present study was proposed with the following two objectives: (i) to undertake phenotypic and molecular characterization of Aspergillus section Flavi isolates recovered from clinical and environmental sources in Kuwait and (ii) to determine antifungal susceptibility profiles by Etest against six antifungal agents, namely, amphotericin B (AP), voriconazole (VO), posaconazole (POS), anidulafungin (AND), micafungin (MYC) and caspofungin (CS). Phenotypic characteristics such as colony and microscopic morphology, sclerotia formation, and production of mycotoxins and aflatoxins were studied. Molecular characterization of selected isolates was based on DNA sequencing of ITS region of rDNA and partial sequencing of -tubulin and calmodulin gene fragments with/without sequencing of additional loci. All 100 isolates were conformable with known colony and microscopic characteristics of A. flavus. Sclerotia were produced by 50 (42 L-phenotype) isolates, typical orange-yellow pigment on Aspertgillus flavus and parasiticus agar was produced by 94 isolates, and 10 isolates showed aflatoxin production. DNA sequencing of multiple loci (acetamidase and O-methyltransferase) identified all isolates as A. flavus complex members within A. flavus clade of Aspergillus section Flavi. Neighbor-joining phylogenetic tree based on combined -tubulin and calmodulin gene also identified all isolates as A. flavus strains. The fingerprinting of 48 selected isolates by 3 microsatellite markers showed genotypic heterogeneity and identified majority (34 of 48) of isolates as unique strains. The MIC90 (μg/ml) values on RPMI medium for AP, VO, POS, AND, MYC and CS by Etest were 3, 0.25, 0.25, 0.002, 0.002 and 0.032, respectively. The zone of inhibition ranges for amphotericin B and voriconazole for clinical isolates by disk diffusion testing were 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). There was no evidence that A. flavus isolates in Kuwait are acquiring resistance to azoles or other antifungal agents tested.
Appears in Programs:0520 Microbiology (M.Sc.)

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