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Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing
1Division of Clinical Microbiology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA.
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.
Insights
l-Canavanine glycine bromothymol blue (CGB) agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) reliably differentiate Cryptococcus gattii and Cryptococcus neoformans. CGB agar alone is insufficient, but effective with other tests or D2 LSU sequencing for clinical identification.
Area of Science:
- Medical Mycology
- Clinical Microbiology
- Molecular Diagnostics
Background:
- Cryptococcus neoformans and Cryptococcus gattii are distinct pathogenic fungi with different epidemiological and clinical profiles.
- Differentiating these species is crucial for appropriate patient management and treatment strategies.
- l-Canavanine glycine bromothymol blue (CGB) agar is a potential tool for differentiation, but its clinical utility requires further evaluation.
Purpose of the Study:
- To evaluate the performance of CGB agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) in differentiating Cryptococcus gattii and Cryptococcus neoformans.
- To assess the reliability of these methods in a routine clinical laboratory setting.
- To integrate effective differentiation methods into the laboratory's yeast identification algorithm.
Main Methods:
- Screening of 102 yeast isolates using CGB agar for glycine assimilation.
- Assessment of urease activity and melanin production.
- D2 LSU sequencing for molecular identification and differentiation.
- Comparison of results from CGB agar and D2 LSU sequencing.
Main Results:
- CGB agar correctly identified all 17 Cryptococcus gattii isolates as positive (blue color) and all 54 Cryptococcus neoformans isolates as negative.
- False positive CGB agar results were observed in yeast isolates other than C. gattii, highlighting its limitation as a standalone test.
- D2 LSU sequencing accurately identified and differentiated all tested C. gattii and C. neoformans isolates.
Conclusions:
- CGB agar, when used in conjunction with urea hydrolysis and Niger seed agar, or with D2 LSU sequencing, provides reliable differentiation of C. gattii and C. neoformans.
- D2 LSU sequencing is a highly accurate method for distinguishing these two fungal species.
- The study successfully incorporated CGB agar and D2 LSU sequencing into the clinical laboratory's yeast identification workflow.
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