Rapid identification of candida species by TaqMan PCR

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Authors: M Guiver, K Levi and B A Oppenheim
Date: May 2001
From: Journal of Clinical Pathology(Vol. 54, Issue 5)
Publisher: BMJ Publishing Group Ltd.
Document Type: Article
Length: 3,075 words

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Aim--To develop and evaluate a Taq-Man [TM] polymerase chain reaction (PCR) for the rapid identification and speciation of candida species.

Methods--Species specific primer and probe sets were designed for the identification of Candida albicans, C parapsilosis, C tropicalis, C krusei, C kefyr, and C glabrata from clinical isolates in a 5' exonuclease (TaqMan [TM]) assay. The probes were labelled with three fluorescent dyes to enable differentiation between species when three primer and probe sets were combined in two multiplexes. The specificity of these assays was evaluated against a range of National Collection of Pathogenic Fungi strains, clinical isolates of yeast, bacterial and viral pathogens.

Results--The primer and probe sets have been shown to be 100% specific for their respective species; there was no crossreaction between any set and human DNA, or extracts from other candida species, fungal, bacterial, or viral pathogens tested. Extracts from two clinical isolates, originally identified as C albicans on the basis of germ tube formation, were not amplified by any of the primer and probe sets. These isolates have been putatively re-identified as C dubliniensis after sequencing of the variable internal transcribed spacer region ITS2 and lack of growth at 45[degrees]C.

Conclusion--This TaqMan assay provides a rapid alternative to conventional culture based techniques for the identification and speciation of the most frequently isolated candida species. The simple extraction method followed by TaqMan PCR can identify the six species mentioned in four hours.

(J Clin Pathol 2001;54:362-366)

Keywords: candida identification; Taqman [TM]; polymerase chain reaction

The rise in incidence of opportunistic mucosal and systemic candida infections has been attributed to the increasing population of immunocompromised patients, intensive immunosuppressive treatment regimens, organ transplantation, and the use of broad spectrum antibiotics. [1-3] Long term treatment with amphotericin and fluconazole has led to antifungal drug resistance in Candida albicans and an increase in non-albicans candida infections. Certain yeast species, such as C krusei, are intrinsically resistant to fluconazole, and isolates of C glabrata and C tropicalis may become resistant. [4] Thus, rapid speciation of candida isolates allows appropriate clinical decisions to be made concerning targeted antifungal treatment and its dosage and duration.

The identification of candida isolates to the species level using carbohydrate assimilation protocols [5] can be problematic, although primary isolation using CHROMagar can be used with reasonable specificity for rapid identification. Pan-fungal PCR assays have been evaluated for the detection of a broad spectrum of species, [6,7] as have candida specific primers [8] and C albicans specific and pan-fungal primers in conjunction with species specific probes. [5,9-11]

In this approach, species specific primers and fluorescent probes have been designed for a 5' exonuclease (TaqMan [TM]) assay for the six most clinically important candida species, namely: C albicans, C parapsilosis, C tropicalis, C krusei, C kefyr, and C glabrata. [12] The primer/probe sets can be used individually or combined in two multiplex sets. Species can be differentiated within the multiplex by the use of three spectrally distinct fluorescent reporter dyes (FAM (6-carboxy-fluorescein), TET (tetrachloro-6-carboxy-fluorescein), or VIC [TM]) attached...

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Gale Document Number: GALE|A76937479