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The agents of cryptococcosis  C. neoformans  and  C. gattii  are important agents of meningoencephalitis in immunocompromised and immunocompetent hosts, respectively. They are grouped into eight major molecular types, VNI-VNIV for  C. neoformans  and VGI-VGIV for  C. gattii . These major molecular types differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. To enable a rapid identification of the major molecular types and potential hybrids within the two species specific probes based on the  PLB1  gene in combination with hyperbranched rolling circle amplification (HRCA) were developed. HRCA was applied to 76 cryptococcal strains, 10 strains each representing the 7 haploid major molecular types, 4 VNIII hybrid strains and 2 inter-species hybrid strains. All strains were correctly identified to the major molecular type and or hybrid type using HRCA alone. To increase the sensitivity a semi-nested PCR step was developed, which will enable the identification of the molecular types/hybrids directly from clinical samples, harboring a low copy number of DNA (40 copies). Thus, HRCA based on the  PLB1  locus alone and in combination with a semi-nested PCR showed to be a specific and sensitive methodology, with a great potential to be used on clinical specimens for the direct diagnosis of the agents of cryptococcosis, including hybrid strains, enabling a rapid and patient tailored treatment choice of this disease.

Identification of the Major Molecular Types of Cryptococcus neoformans and C. gattii by Hyperbranched Rolling Circle Amplification