Molecular epidemiology and antifungal susceptibilities of Cryptococcus neoformans in bird droppings from zoological gardens and public places in Peninsular Malaysia

Cryptococcus neoformans, a ubiquitous fungus commonly found in bird droppings, poses a significant health risk by causing cryptococcal meningitis especially in immunocompromised individuals. In Malaysia, clinical reports from 1964-2010 documented an increasing incidence of C. neoformans cases, even in healthy individuals. Nevertheless, studies focusing on C. neoformans occurrence in birds are limited, with the last study conducted in 2005, focusing solely on the Klang Valley region. We aimed to update the molecular epidemiology and antifungal susceptibility of C. neoformans in bird droppings from public areas and zoological gardens across Peninsular Malaysia. Molecular identifications were performed using nested-PCR with CNLAC1 outer and inner primer pairs for the primary and secondary PCR. Antifungal susceptibility tests were conducted against Amphotericin B, Fluconazole, and Itraconazole. The hygiene and environmental conditions of the zoological gardens were recorded. A total of 509 bird droppings were collected: 257 (50.5%) from public places and 252 (49.5%) from zoological gardens. Most samples from public areas were from common pigeons (n = 144; 56.0%), while samples from Mandarin ducks predominated in zoological gardens (n = 40; 15.9%). The overall prevalence of C. neoformans was 42.6% (217/509), with a higher prevalence in zoological gardens (116/252; 46.0%) versus public places (101/257; 39.3%) (P = 0.125). Notably, common pigeons in zoological gardens showed a significantly higher carrier rate (80.8%) versus in public places (54.9%) (P = 0.013). Other species with high carrier rates in zoological garden included Indian peafowls (61.9%) and budgie birds (61.3%). In public areas, apart from pigeons; doves (43.5%) also exhibited a high prevalence. Enclosure density and Columbidae family were found to be associated with high positivity rate in zoological gardens. Two strains were identified: C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A). One isolate exhibited resistance to Itraconazole. This study highlights the need for ongoing public health surveillance and preventive measures particularly in settings where human-bird interactions are frequent.