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Published on: September 5, 2013
Cryptococcus species identification by multiplex PCR
Ana Lusia Leal1, Josiane Faganello, Maria Cristina Bassanesi
1Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
Abstract:
Members of the Cryptococcus species complex are encapsulated basidiomycetous yeasts, which can affect the central nervous system (CNS) and if untreated may cause meningitis. Cryptococcus neoformans is an opportunistic pathogen causing infections mainly in immunocompromised individuals. Cryptococcus gattii is a primary pathogen responsible for a high incidence of cryptococcomas in the lung and brain and shows a delayed response to antifungal therapy. The differentiation between the two species is primarily based on their growth on and color change of canavanine - glycine-bromothymol blue agar (CGB). Since this test is not always reliable, a multiplex PCR to identify both Cryptococcus species using more than 130 samples was standardized and the results obtained compared to those with the CGB test, using the Crypto Check serotyping kit as the standard. The multiplex PCR was shown to be more specific than the CGB test, in that results obtained with it were in agreement with those from serotyping all the samples, while the data from the CGB test disagreed with 6 out of 131 samples.
Insights
Differentiating Cryptococcus neoformans and Cryptococcus gattii is crucial for patient care. Multiplex PCR offers a more accurate method than the traditional CGB test for identifying these fungal pathogens.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- The Cryptococcus species complex comprises encapsulated yeasts that can cause severe central nervous system infections, including meningitis.
- Cryptococcus neoformans is an opportunistic pathogen, primarily affecting immunocompromised individuals.
- Cryptococcus gattii is a primary pathogen causing lung and brain cryptococcomas, often with delayed antifungal treatment response.
Purpose of the Study:
- To develop and validate a multiplex PCR assay for accurate differentiation of Cryptococcus neoformans and Cryptococcus gattii.
- To compare the diagnostic performance of the multiplex PCR assay against the conventional Canavanine-Glycine-Bromothymol blue (CGB) agar test.
Main Methods:
- Standardization of a multiplex PCR assay using over 130 clinical samples.
- Comparative analysis of multiplex PCR results against the CGB agar test and a Crypto Check serotyping kit as the gold standard.
Main Results:
- The multiplex PCR assay demonstrated higher specificity compared to the CGB agar test.
- Multiplex PCR results showed complete agreement with the serotyping standard for all samples.
- The CGB agar test yielded discrepant results for 6 out of 131 samples when compared to serotyping.
Conclusions:
- Multiplex PCR is a more reliable and specific method for differentiating Cryptococcus neoformans and Cryptococcus gattii than the CGB agar test.
- Accurate species identification is essential for appropriate clinical management of cryptococcosis.
