Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes

and are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal anti...

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Bibliographic Details
Published in:Journal of fungi (Basel) 2022-04, Vol.8 (5), p.462
Main Authors: Tay, Enoch, Chen, Sharon C-A, Green, Wendy, Lopez, Ronald, Halliday, Catriona L
Format: Article
Language:English
Subjects:
Cryptococcosis
Cryptococcus
Cryptococcus gattii
Cryptococcus neoformans
Cryptococcus PCR
Cytochrome
Cytochrome b
diagnosis of cryptococcosis
DNA sequencing
Epidemiology
Genotypes
Laboratories
Microscopy
Mitochondria
Paraffin
Pathogens
Polymerase chain reaction
Species
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Summary:and are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome ( ) gene to detect and in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The -directed assay accurately detected and identified all eight / genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The -specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.
ISSN:2309-608X
2309-608X
DOI:10.3390/jof8050462