Molecular epidemiology and antifungal susceptibility of Cryptococcus neoformans isolated from bird droppings in selected public places and zoological gardens of peninsular Malaysia

Cryptococcus neoformans is a ubiquitous fungus commonly associated with pigeon droppings. This organism poses a significant health risk, causing cryptococcosis in both immunocompromised and immunocompetent individuals. In Malaysia, clinical reports spanning 1953 to 2020 indicate a rising incidence of cryptococcal infections especially cerebral cryptococcosis. However, research on the occurrence of C. neoformans in bird hosts remains scarce, with the last study conducted in 2005 and restricted to the Klang Valley region. This study aimed to provide updated data on the molecular epidemiology of C. neoformans in bird droppings by encompassing its prevalence, associated risk factor in zoological garden, antifungal susceptibility and serotypes from samples collected in public places and zoological gardens across Peninsular Malaysia. Samples were first cultured on Bird Seed Agar (BSA) for presumptive identification based on melanin pigment production. Nested PCR employing CNLAC1 gene-specific outer and inner primers was performed for molecular identification. Susceptibility of the positive isolates to antifungals Amphotericin B, Fluconazole and Itraconazole was assessed. Bird species, hygiene and environmental aspects of the zoological gardens were documented. Overall, 509 bird droppings were collected: public places [n=257 (50.5%)] and zoological gardens [n=252 (49.5%)]. Common pigeons were the predominant samples collected from public places (n = 144; 56.0%). Mandarin ducks represented the dominant source of samples from zoological gardens (n = 40; 15.9%). Common pigeons in zoological gardens exhibited a significantly higher positivity rate (80.8%) compared to the common pigeon in public places (54.9%) (P = 0.013). High positivity rates in zoological gardens were observed in Indian peafowls (61.9%) and budgies (61.3%). Doves in this study exhibited a high positivity rate (43.5%) in public places. The positivity rate of C. neoformans in zoological gardens were found to be associated by enclosure densities (P=0.02) and the Columbidae family (P= 0.002). Only one isolate showed resistance to Itraconazole, while another exhibited intermediate resistance to Fluconazole. Two serotypes in this study were identified, serotype A (C. neoformans var. grubii) and serotype D (C. neoformans var. neoformans). These findings reveal markedly high C. neoformans positivity rate, most notably in common pigeons but also in other bird species, with enclosure density in zoological gardens further contributing to colonization. The detection of clinically relevant serotypes and potential resistance to Itraconazole also highlighted the importance of continuous public health monitoring and the implementation of preventive strategies, especially in environments with frequent human-bird interactions.