644. Evaluation of a Multiplex PCR for Typing of Cryptococcus Spp
Session: Abstracts: Mycology
Friday, October 22, 2010
Background: Cryptococcus gattii and C. neoformans are both endemic in British Columbia making diagnosis a challenge due to the similarities between these two species.  To improve the turn around time for clinical diagnosis and enhance public health surveillance and monitoring, identification of C. gattii in B.C using a multiplex PCR with typing properties was evaluated against pre-established Restriction Fragment Length Polymorphism (RFLP) testing and Multi-locus Sequence Typing (MLST) data.

Methods: We selected 107 C. gattii isolates from clinical, veterinarian and environmental sources for the multiplex (LAC1/CAP64) PCR study. The previously validated URA5 gene RFLP assay as well as MLST of partial fragments of 7 housekeeping genes (URA5, IGS1, PLB1, CAP59, LAC1, SOD1, and GPD1) were also performed on all study samples. In addition, 13 clinical C. neoformans isolates were subjected to the multiplex PCR, to demonstrate species-level discrimination. A cost evaluation was performed in order to determine the most efficient scheme for diagnostic laboratory work flow.

Results: There was a perfect correlation between the multiplex PCR and RFLP results. For C. gattii, three PCR types were obtained, B1, B2 and C, corresponding to RFLP types VGI, VGII, and VGIII, respectively. These findings also matched the MLST results. C. neoformans isolates were separated into three additional PCR types, A, AD, and D, corresponding to RFLP types VNI/II, VNIII, and VNIV.

Conclusion: The multiplex PCR is accurate in identification of C. gattii and provides a test turn around time that is one day shorter with a slight reduction in cost in comparison to RFLP, and is significantly cheaper and faster than MLST.  This multiplex PCR is therefore suitable for routine diagnosis of Cryptococcus species for clinical management.


Subject Category: M. Mycology including clinical and basic studies of fungal infections

Speakers:
Andrew Balbirnie, BS , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada
Danielle Jorgensen, BS , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada
Loretta Janz, BS , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada
Min-Kuang Lee, MS , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada
Sultana Mithani, BS , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada
Muhammad G. Morshed, PhD , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada
Linda MN Hoang, MD, FRCPC , BCCDC Public Health Microbiology and Reference Laboratory, Provincial Health Services Authority, Vancouver, BC, Canada

Disclosures:

A. Balbirnie, None

D. Jorgensen, None

L. Janz, None

M. K. Lee, None

S. Mithani, None

M. G. Morshed, None

L. M. Hoang, None

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