A Built-in Multiplex Protocol to Identify the Opportunistic Fungal Pathogen Candida glabrata

Ref ID: 18782

Author:

A. De Las Peñas, PhD – Professor, D. Barron, MS – Research Assistant, M. Cuellar, PhD – Professor, I. Castaño, PhD – Professor;

Author address:

Instituto Potosino de Investigacion Cientifica y Tecnologica, San Luis Potosi, San Luis Potosi, Mexico.

Full conference title:

52nd Annual ICAAC

Date: 9 September 2014

Abstract:

Background: Candida glabrata is a commensal yeast that acts as an opportunistic pathogen of inmunocompromised patients. C. glabrata has emerged as the second cause of mucosal, urinary tract and bloodstream fungal infections; 18-26% of all Candida systemic infections in the US. C. glabrata infections are common in patients with diabetes, cancer, and extensive use of antibiotics. C. glabrata infections are associated with high mortality rates and are difficult to treat, since C. glabrata is naturally less susceptible to fluconazole. Thus a rapid and accurate diagnosis is of outmost importance: first to determine the presence of C. glabrata and second, to determine which antifungal to use. There are 3 general diagnostic methods for the identification of C. glabrata: a) selection on specific media and morphology, b) carbohydrate assimilation, and c) molecular methods that include: PCR amplification of the ITS2 variable regions and sequencing, fluorescence in situ hybridization (FISH), identification of specific sequences in particular genes, and detection of specific proteins. It has been shown recently that both C. glabrata clinical isolates and laboratory reference strains, have a high genomic variability and different phenotypes regarding resistance to fluconazole, biofilm formation, and resistance to oxidative stress. This genomic variability has to be taken into consideration when applying single target PCR for diagnostics. Methods: The protocol identifies C. glabrata directly from 1 mL urine and pre-cultured blood samples from hospitalized patients. Our method is based on a multiplex, single PCR reaction of a specific region that is present 9 times throughout the C. glabrata genome. Results: In this work, we present a molecular protocol that identifies 100% of C. glabrata isolates identified by the gold standard method. Conclusions: This method is extremely specific, since PCR reactions with genomic DNA mixtures from different but phylogenetically related yeast, do not amplify. This strategy has not been addressed in any prior report, it is novel, not obvious and subject to industrial application. We have patents protecting the genomic regions of interest.

Abstract Number: M-1668

Conference Poster: y

Conference Year: 2012

Link to conference website: NULL

New link: NULL

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