Trilles Luciana, Wang Bin, Firacative Carolina, Lazéra Márcia Dos Santos, Wanke Bodo, Meyer Wieland
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.
Westmead Millennium Institute, University of Sydney, Retroviral Genetic Laboratory, Centre for Virus Research, Sydney, Australia.
PLoS One. 2014 Apr 15;9(4):e94648. doi: 10.1371/journal.pone.0094648. eCollection 2014.
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.
新型隐球菌和格特隐球菌是隐球菌病的病原体，分别是免疫功能低下和免疫功能正常宿主中脑膜脑炎的重要病原体。它们被分为八大主要分子类型，新型隐球菌为VNI-VNIV，格特隐球菌为VGI-VGIV。这些主要分子类型在宿主范围、流行病学、抗真菌药敏性和地理分布方面存在差异。为了能够快速鉴定这两个物种内的主要分子类型和潜在杂交种，基于PLB1基因结合超分支滚环扩增（HRCA）开发了特异性探针。HRCA应用于76株隐球菌菌株，其中10株分别代表7种单倍体主要分子类型，4株VNIII杂交菌株和2株种间杂交菌株。仅使用HRCA就可将所有菌株正确鉴定到主要分子类型和/或杂交类型。为了提高灵敏度，开发了一个半巢式PCR步骤，这将能够直接从临床样本中鉴定分子类型/杂交种，这些样本中DNA拷贝数较低（40个拷贝）。因此，基于PLB1基因座单独使用以及与半巢式PCR结合使用的HRCA显示是一种特异性和灵敏的方法，在用于隐球菌病病原体（包括杂交菌株）直接诊断的临床标本上具有很大的应用潜力，能够实现对该疾病的快速且针对患者的治疗选择。
