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Journal of Clinical Microbiology, November 2009, p. 3669-3672, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.01072-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Cryptococcus gattii by Use of L-Canavanine Glycine Bromothymol Blue Medium and DNA Sequencing{triangledown}

K. R. Klein, L. Hall, S. M. Deml, J. M. Rysavy, S. L. Wohlfiel, and N. L. Wengenack*

Division of Clinical Microbiology, Mayo Clinic, 200 First St. SW, Rochester, Minnesota 55905

Received 2 June 2009/ Returned for modification 29 July 2009/ Accepted 21 September 2009

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi. Cryptococcus neoformans is ecologically widespread and affects primarily immunocompromised patients, while C. gattii is traditionally found in tropical climates and has been reported to cause disease in immunocompetent patients. L-Canavanine glycine bromothymol blue (CGB) agar can be used to differentiate C. neoformans and C. gattii, but there are few reports of its performance in routine clinical practice. Growth of C. gattii on CGB agar produces a blue color, indicating the assimilation of glycine, while C. neoformans fails to cause a color change. Using reference and clinical strains, we evaluated the ability of CGB agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) to differentiate C. neoformans and C. gattii. One hundred two yeast isolates were screened for urease activity, melanin production, and glycine assimilation on CGB agar as well as by D2 sequencing. Seventeen of 17 (100%) C. gattii isolates were CGB positive, and 54 of 54 C. neoformans isolates were CGB negative. Several yeast isolates other than the C. gattii isolates were CGB agar positive, indicating that CGB agar cannot be used alone for identification of C. gattii. D2 correctly identified and differentiated all C. gattii and C. neoformans isolates. This study demonstrates that the use of CGB agar, in conjunction with urea hydrolysis and Niger seed agar, or D2 LSU sequencing can be reliably used in the clinical laboratory to distinguish C. gattii from C. neoformans. We describe how CGB agar and D2 sequencing have been incorporated into the yeast identification algorithm in our laboratory.

* Corresponding author. Mailing address: Division of Clinical Microbiology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Phone: (507) 266-4822. Fax: (507) 284-9859. E-mail: wengenack.nancy{at}mayo.edu

{triangledown} Published ahead of print on 30 September 2009.

Journal of Clinical Microbiology, November 2009, p. 3669-3672, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.01072-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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