A quick and low-cost PCR-based assay for Candida spp. identification in positive blood culture bottles

A quick and low-cost PCR-based assay for Candida spp. identification in positive blood culture bottles

Author Xafranski, Hemilio Autor UNIFESP Google Scholar
Melo, Analy Salles de Azevedo Autor UNIFESP Google Scholar
Machado, Antonia M. Autor UNIFESP Google Scholar
Briones, Marcelo Ribeiro da Silva Autor UNIFESP Google Scholar
Colombo, Arnaldo Lopes Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract Background: Differences in the susceptibility of Candida species to antifungal drugs make identification to the species level important for clinical management of candidemia. Molecular tests are not yet standardized or available in most clinical laboratories, although such tests can reduce the time required for species identification, as compared to the conventional culture-based methods. To decrease laboratory costs and improve diagnostic accuracy, different molecular methods have been proposed, including DNA extraction protocols to produce pure DNA free of PCR inhibitors. the objective of this study was to validate a new format of molecular method, based on the internal transcribed spacer (ITS) of the rDNA gene amplification followed by sequencing, to identify common and cryptic Candida species causing candidemia by analyzing DNA in blood culture bottles positive for yeasts.Methods: for DNA extraction, an in-house protocol based on organic solvent extraction was tested. Additional steps of liquid nitrogen incubation followed by mechanical disruption ensured complete cell lysis, and highly pure DNA. One hundred sixty blood culture bottles positive for yeasts were processed. PCR assays amplified the ITS region. the DNA fragments of 152 samples were sequenced and these sequences were identified using the GenBank database (NCBI). Molecular yeast identification was compared to results attained by conventional method.Results: the organic solvent extraction protocol showed high reproducibility in regards to DNA quantity, as well as high PCR sensitivity (10 pg of C. albicans DNA and 95% amplification on PCR). the identification of species at the molecular level showed 97% concordance with the conventional culturing method. the molecular method tested in the present study also allowed identification of species not commonly implicated in human infections.Conclusions: This study demonstrated that our molecular method presents significant advantages over the conventional yeast culture identification method by providing accurate results within 24 hours, in contrast to at least 72 hours required by the automated conventional culture method. Additionally, our molecular method allowed the identification of mixed infections, as well as infections due to emergent fungal pathogens. This economical DNA extraction method developed in our laboratory provided high-quality DNA and 60% cost savings compared to commercial methods.
Keywords Candidemia
Molecular diagnosis
ITS region sequencing
Fungal DNA extraction
Language English
Date 2013-10-07
Published in Bmc Infectious Diseases. London: Biomed Central Ltd, v. 13, 7 p., 2013.
ISSN 1471-2334 (Sherpa/Romeo, impact factor)
Publisher Biomed Central Ltd
Extent 7
Origin http://dx.doi.org/10.1186/1471-2334-13-467
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000328900200003
URI http://repositorio.unifesp.br/handle/11600/36869

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