Direct detection of fungus from clinical specimens by Pyrosequence analysis

• Main Authors: Ching Yi Chuang, 莊靜怡
• Other Authors: T. L. Wu
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/72256415740292717624

碩士 === 長庚大學 === 醫學生物技術暨檢驗學系 === 98 === Fungal blood stream infection is a kind of invasive fungal infection(IFI). Patients who suffering from immunodeficiency or immunocompromise and had IFIs accompanied high mortalities and morbidities. Previous studies showed that delaying treatment in greater than 12 hours after positive results from blood samples, mortality of candidiasis increased from 10% to 30%. While traditional culture system has a poor sensitivity, and takes longer period in identification, methods of high throughput and more accuracy for diagnosis of fungal infection are urgently needed. In this study, we amplified ITS 1 of fungal rDNA by panfungal PCR primer and sequencing the PCR products with a specially designed 22 nucleotides degenerative sequencing primer-5.8R at the 5' of the 5.8 rDNA. Medical important fungal pathogens including 8 genera and 17 species, which contains Candida (8 species), Aspergillus (5 species), Cryptococcus neoformans, Fusaium solani, Scedosporium prolificans, and Penicillium marneffei. 12/15(80%) of reference strains identified to the species level and 2/15 (13.3%) of strains identified to the genus level. 57/89 (64.0%) of clinical isolates were consisted with the results of conventional identification. There are 10/14(71.4%) of clinical isolates which identified to the genus level by traditional methods, but are now identified to the species level by using pyrosequencing analysis. 63/66 (95.5%) of positive blood culture bottles can be identified to the species level. That all the specimens above can be identified to genera with the exception of A. versicolor. The detection limit of pyrosequencing are 1 pg/μl of C. albicans genomic DNA and 200 CFU/ml of microbial spiked whole blood. Suggested the prospects of pyrosequencing for directly identify fungal pathogens from clinical specimens. This data implicated pyrosequencing is a potential tool for clinical applications.