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Abstract Cryptococcosis is among the most severe mycotic diseases caused by basidiomycetous encapsulated yeasts C. neoformans and C. gattii. It is a world-wide life-threatening opportunistic fungal infection affecting human and animals. Cryptococcus neoformans and C. gattii show different and overlapping ecological niches. The present study was conducted to shed light on occurance of C. neoformans and C. gattii from environmental samples from different localities in Egypt, as well as molecular methods of identification of Cryptococcus species. Totally, 70 pigeon droppings, 50 captive bird droppings and 80 Eucalyptus tree samples were collected from multiple localities for isolatation of C. neoformans and C. gattii. The recovered isolates were identified by both phenotypic and molecular methods. Yeasts were primary isolated on SDA media and the suspected ones were subjected to phenotypic identification for detecticton of the main characteristics of Cryptococcus species, as capsule production, growth at 37°C, and hydrolysis of urea. Moreover, C. gattii was differentiated from C. neoformans via the capability of using glycine as carbone and nitrogen source and assimilation of D-tryptophan using Cryptococcus differential agar media. Only 4 isolates showed brown mucoid colonies and were identified as C. gattii. Meanwhile, the 23 remaining isolates showed light blue dry colonies and identified as C. neoformans. Multiplex PCR was employed for identification of Cryptococcus species using species-specific primers CNa-70S, CNa-70A for C. |