Molecular characterization of Cryptococcus neoformans and Cryptococcus gattii from environmental sources and genetic comparison with clinical isolates in Apulia, Italy.

The present study investigated the environmental distribution of Cryptococcus neoformans and C. gattii species complex molecular types, mating types and sequence types in Apulia, a region of Southern Italy. A total of 2078 specimens from arboreal and animal sources were analyzed. The percentage of positive samples was similar among both arboreal and animal specimens: 4.2% vs. 5.1% for C. neoformans species complex and 0.6% vs. 1.4% for C. gattii species complex. Molecular typing identified 78 isolates as VNI (76 αA and two aA), one as AD-hybrid αADa, and 16 as VGI aB. VNI isolates presented 10 different sequence types (STs) and VGI isolates two. The most frequent STs among C. neoformans and C. gattii species complex isolates were ST23 (51%) and ST156 (90%), respectively. Comparison with molecular types and STs results obtained from 21 clinical isolates collected in Apulia showed that one C. neoformans VNI clinical isolate shared an identical sequence type of one arboreal isolate (ST61) and that one C. gattii VGI clinical isolate matched with the main ST (ST156) present in the environment. In addition, molecular type VNIV was found only among clinical isolates and was absent in the investigated environmental area. In conclusion, the present study identified which C. neoformans and C. gattii species complex genotypes are circulating in Apulia, defined their ecological niches and revealed the relationship with clinical cases. It represents a basal study for addressing future investigations and public health interventions in the region.

Cryptococcus gattii sero-mating type allelic pattern determined by multiplex PCR.

Molecular methods to differentiate serotypes, mating types and molecular types of Cryptococcus neoformans and C. gattii are important tools to understand epidemiology and pathogenesis of these pathogens. In this study, a multiplex polymerase chain reaction (PCR) approach was applied to sero-mating typing of C. gattii strains. Four pairs of primers were designed to target 4 allele-specific genes located in the mating-type locus. Twenty-three C. gattii strains, presenting different mating types and serotypes, were tested to validate the method. The method was able to identify all sero-mating allelic patterns including hybrid combinations, and therefore, it represents a simple one-step PCR for sero-mating typing of C. gattii strains.