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Updated: May 1, 2026
Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans
Published on: March 19, 2015
Identification of the major molecular types of Cryptococcus neoformans and C. gattii by Hyperbranched rolling circle
Luciana Trilles1, Bin Wang2, Carolina Firacative3
1Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Sydney Medical School - Westmead Hospital, Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Westmead Millennium Institute for Infectious Diseases and Biosecurity, Sydney, Australia; Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Laboratório de Micologia, Rio de Janeiro, Brazil.
Abstract:
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.
Insights
Rapid identification of Cryptococcus neoformans and Cryptococcus gattii molecular types is now possible. A new method using hyperbranched rolling circle amplification (HRCA) accurately detects fungal strains and hybrids for tailored treatment.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Cryptococcosis is caused by Cryptococcus neoformans and Cryptococcus gattii, leading to meningoencephalitis in various hosts.
- These fungal species are classified into eight major molecular types (VNI-VNIV for C. neoformans, VGI-VGIV for C. gattii) and hybrids, which exhibit distinct epidemiological and clinical characteristics.
- Accurate molecular typing is crucial for understanding disease transmission and guiding patient treatment.
Purpose of the Study:
- To develop a rapid and specific method for identifying the major molecular types and hybrid strains of Cryptococcus neoformans and Cryptococcus gattii.
- To evaluate the efficacy of a novel diagnostic approach for cryptococcosis agents.
Main Methods:
- Development of specific probes targeting the PLB1 gene.
- Application of hyperbranched rolling circle amplification (HRCA) for initial strain identification.
- Incorporation of a semi-nested PCR step to enhance sensitivity for low DNA quantities (down to 40 copies).
Main Results:
- HRCA alone correctly identified all 76 tested cryptococcal strains to their major molecular or hybrid type.
- The combined HRCA and semi-nested PCR method demonstrated high specificity and sensitivity.
- The developed methodology successfully identified low DNA copy numbers, indicating potential for clinical sample analysis.
Conclusions:
- HRCA based on the PLB1 locus, with or without semi-nested PCR, is a specific and sensitive diagnostic tool.
- This method holds significant potential for direct diagnosis of cryptococcosis agents, including hybrid strains, from clinical specimens.
- Enables rapid and personalized treatment strategies for patients with cryptococcosis.
