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.
journal_name
BMC Infect Disjournal_title
BMC infectious diseasesauthors
Xafranski H,Melo AS,Machado AM,Briones MR,Colombo ALdoi
10.1186/1471-2334-13-467subject
Has Abstractpub_date
2013-10-07 00:00:00pages
467issn
1471-2334pii
1471-2334-13-467journal_volume
13pub_type
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pub_type: 杂志文章,多中心研究
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更新日期:2020-11-23 00:00:00
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更新日期:2013-08-27 00:00:00
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pub_type: 杂志文章
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journal_title:BMC infectious diseases
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journal_title:BMC infectious diseases
pub_type: 杂志文章
doi:10.1186/s12879-018-3596-5
更新日期:2018-12-18 00:00:00
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更新日期:2013-09-05 00:00:00