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IDENTIFICATION OF THE MAJOR MOLECULAR TYPES OF CRYPTOCOCCUS NEOFORMANS AND C. GATTII BY HYPERBRANCHED ROLLING CIRCLE AMPLIFICATION
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Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil / The University of Sydney. Marie Bashir Institute for Infectious Diseases and Biosecurity. Sydney Medical School. Centre for Infectious Diseases and Microbiology. Molecular Mycology Research Laboratory. Sydney, Australia.
University of Sydney. Westmead Millennium Institute. Centre for Virus Research, Retroviral Genetic Laboratory. Sydney, Australia.
The University of Sydney. Marie Bashir Institute for Infectious Diseases and Biosecurity. Sydney Medical School. Centre for Infectious Diseases and Microbiology. Molecular Mycology Research Laboratory. Sydney, Australia.
Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.
The University of Sydney. Marie Bashir Institute for Infectious Diseases and Biosecurity. Sydney Medical School. Centre for Infectious Diseases and Microbiology. Molecular Mycology Research Laboratory. Sydney, Australia.
University of Sydney. Westmead Millennium Institute. Centre for Virus Research, Retroviral Genetic Laboratory. Sydney, Australia.
The University of Sydney. Marie Bashir Institute for Infectious Diseases and Biosecurity. Sydney Medical School. Centre for Infectious Diseases and Microbiology. Molecular Mycology Research Laboratory. Sydney, Australia.
Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Micologia. Rio de Janeiro, RJ, Brasil.
The University of Sydney. Marie Bashir Institute for Infectious Diseases and Biosecurity. Sydney Medical School. Centre for Infectious Diseases and Microbiology. Molecular Mycology Research Laboratory. Sydney, Australia.
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.
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