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Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing.
J Clin Microbiol. 2009 Nov; 47(11):3669-72.JC

Abstract

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.

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Authors+Show Affiliations

Klein KR
Division of Clinical Microbiology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA.
Hall L
No affiliation info available
Deml SM
No affiliation info available
Rysavy JM
No affiliation info available
Wohlfiel SL
No affiliation info available
Wengenack NL
No affiliation info available

MeSH

AlgorithmsBromthymol BlueCanavanineClinical Laboratory TechniquesCryptococcosisCryptococcus gattiiCryptococcus neoformansCulture MediaDNA, FungalDNA, RibosomalDiagnosis, DifferentialFungal ProteinsGlycineHumansMelaninsRNA, Ribosomal, 28SSequence Analysis, DNAUrease

Pub Type(s)

Evaluation Study
Journal Article

Language

eng

PubMed ID

19794048

Citation

Klein, K R., et al. "Identification of Cryptococcus Gattii By Use of L-canavanine Glycine Bromothymol Blue Medium and DNA Sequencing." Journal of Clinical Microbiology, vol. 47, no. 11, 2009, pp. 3669-72.
Klein KR, Hall L, Deml SM, et al. Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing. J Clin Microbiol. 2009;47(11):3669-72.
Klein, K. R., Hall, L., Deml, S. M., Rysavy, J. M., Wohlfiel, S. L., & Wengenack, N. L. (2009). Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing. Journal of Clinical Microbiology, 47(11), 3669-72. https://doi.org/10.1128/JCM.01072-09
Klein KR, et al. Identification of Cryptococcus Gattii By Use of L-canavanine Glycine Bromothymol Blue Medium and DNA Sequencing. J Clin Microbiol. 2009;47(11):3669-72. PubMed PMID: 19794048.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing. AU - Klein,K R, AU - Hall,L, AU - Deml,S M, AU - Rysavy,J M, AU - Wohlfiel,S L, AU - Wengenack,N L, Y1 - 2009/09/30/ PY - 2009/10/2/entrez PY - 2009/10/2/pubmed PY - 2010/1/8/medline SP - 3669 EP - 72 JF - Journal of clinical microbiology JO - J Clin Microbiol VL - 47 IS - 11 N2 - 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. SN - 1098-660X UR - https://www.unboundmedicine.com/medline/citation/19794048/Identification_of_Cryptococcus_gattii_by_use_of_L_canavanine_glycine_bromothymol_blue_medium_and_DNA_sequencing_ L2 - https://journals.asm.org/doi/10.1128/JCM.01072-09?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -