The Cryptococcus neoformans/Cryptococcus gattii species complex in Asia

For decades etiologic agents of cryptococcosis were named C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. Nowadays, based on the combination of various speciation criteria, including ecological, geographical, genetic, phenetic and phylogenetic characteristics, C. neoformans var. grubii and C. neoformans var. neoformans are recognized as C. neoformans and C. deneoformans, and C. gattii is divided into 5 species, i.e. C. gattii, C. bacillisporus, C. deuterogattii, C. tettragattii and C. decagattii. Most cases of cryptococcal meningitis are caused by C. neoformans, with most patients (>90%) occurring in Sub-Saharan Africa and the Asia-Pacific region. Using MLST analysis the global genetic diversity of C. neoformans showed that isolates from Africa had a high genetic diversity, while isolates from Asia, particular East-Southeast-South Asia, had a more limited genetic diversity. Population structure analysis of C. neoformans in Asia showed that each genotype (sequence type) had an unique distribution at each locale. In addition, microsatellite analysis showed that each genotype or microsatellite complex had a limited geographic occurrence. Ancestral reconstruction using Bayesian Binary Markov Chain Monte Carlo analysis confirmed that Africa is a possible place of origin of C. neoformans before spreading globally. In contrast, S-DIVA supported the “Continental drift” hypothesis that C. neoformans originated on Pangea before moving to the modern continents.
In our study, 5-fluorocytosine-, fluconazole- and double (5-fluorocytosine-fluconazole and fluconazole-voriconazole)-resistant isolates of C. neoformans were found. It is common that 5-fluorocytosine and azole resistant isolates occur among clinical isolates. However, the presence of isolates that were intrinsically resistant to 5-fluorocytosine in Indonesia and Thailand is a concern when this antifungal drug will become available for therapy of cryptococcosis in these countries.
Cryptococcus gattii was isolated for the first time from soil and debris samples collected from a hollow of a Castanopsis argyrophylla tree in Chiang Mai, Thailand. This isolate was typed as AFLP4/VGI and, hence, represented C. gattii sensu stricto. The isolation of members of the C. neoformans/C. gattii species complex is performed by culturing on selective and differential media, such as caffeic acid agar or Niger seed agar. These culture media induce melanization of yeasts of the C. neoformans/C. gattii species complex, resulting in brown colonies that differ from most other yeasts. Based on this melanization process of these yeasts, a novel Banana blossom agar was formulated for isolation of members of the C. neoformans/C. gattii species complex. This medium may find use in resource limited settings as banana trees occur widely in areas where many cryptococcosis patients live.