title: Identification of the major molecular types of Cryptococcus neoformans and C. gattii by Hyperbranched rolling circle amplification.
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.
authors.affiliation: Molecular 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.
authors.forename: Luciana
authors.initials: L
authors.lastname: Trilles
authors.affiliation: Westmead Millennium Institute, University of Sydney, Retroviral Genetic Laboratory, Centre for Virus Research, Sydney, Australia.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: Molecular 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.
authors.forename: Carolina
authors.initials: C
authors.lastname: Firacative
authors.affiliation: Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Laboratório de Micologia, Rio de Janeiro, Brazil.
authors.forename: Márcia Dos Santos
authors.initials: Mdos S
authors.lastname: Lazéra
authors.affiliation: Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Laboratório de Micologia, Rio de Janeiro, Brazil.
authors.forename: Bodo
authors.initials: B
authors.lastname: Wanke
authors.affiliation: Molecular 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.
authors.forename: Wieland
authors.initials: W
authors.lastname: Meyer
mesh_terms: D003455:Cryptococcus neoformans / Q000145:classification* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D015345:Oligonucleotide Probes
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