Multiplex PCR - RFLP Assay for Identification of Cryptococcus neoformans and Cryptococcus gattii Isolated from Birds’ Droppings and Eucalyptus Trees

Abstract

Cryptococcus neoformans/ Cryptococcus gattii (C. neoformans/ C. gattii) species complex are encapsulated basidiomycetous yeasts, causing cryptococcosis which is a life-threatening fungal disease of the pulmonary and central nervous system of humans and animals. This study aimed to investigate the recovery rate of C. neoformans and C. gattii from bird droppings and Eucalyptus trees in Egypt as well as the performance of the phenotypic and molecular identification methods for Cryptococcus species identification. Overall, 27 Cryptococcus isolates (13.5%) were isolated from 200 examined samples including 70 pigeon droppings, 50 captive birds' droppings, and 80 Eucalyptus trees samples. The recovered isolates were phenotypically identified based on macro-and micro-morphological characters, urease test, and differentiation using cryptococcus differential agar media. Multiplex polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis using EcoRI restriction enzyme for confirmation of species identification. The molecular methods identified 20 C. neoformans from pigeon's droppings (12/70, 17.14%) and captive birds' droppings samples (8/50, 16%), as well as 7 C. gattii from Eucalyptus trees (7/80, 8.75%). Molecular identification results did not correspond with those of the phenotypic identification methods in three isolates (11.11%), as phenotypic methods identified only 4 C. gattii isolates and molecular methods identified 7 isolates. In conclusion, multiplex PCR and RFLP analysis of multiplex PCR products are rapid, sensitive, species-specific, and more reliable methods for identification of Cryptococcus species and may be used as a complementary to phenotypic methods to avoid false-negative and falsepositive results.