Visual Analysis of DNA Microarray Data for Accurate Molecular Identification of Non-albicans Candida Isolates from Patients with Candidemia Episodes

Abstract

The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance. Candida species are the predominant cause of systemic fungal infections in hospitalized patients and represent the fourth most common microorganisms found in blood cultures (1, 2). In a large, laboratory-based study conducted in Brazilian hospitals, the incidence of candidemia was found to be 2.49 cases per 1,000 patient admissions (3), which is higher than that in the United States (0.28/1,000), Europe (0.2/1,000), or France (0.17/1,000) (4-7). In our setting, the reported candidemia incidence was 0.54 cases per 1,000 patient days, with 56% non-albicans Candida spp. (8). An increase in the proportion of non-albicans Candida spp. has been observed worldwide with distinctive patterns of antifungal susceptibility (9, 10).The development of automated microbiology systems has represented a significant improvement in the identification of Candida spp. However, their diagnostic accuracy has been reported to range from 43 to 95% (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Considering the frequency of occurrence, the high mortality rate, and the susceptibility patterns of non-albicans Candida spp., novel point-of-care molecular technologies are urgently needed in clinical practice. Our study evaluated the ability of a novel plastic slide-based DNA microarray to identify non-albicans Candida spp. isolated from blood cultures.(Partial results of this study were presented at the 18th Congress of the International Society for Human and Animal Mycology, Berlin, Germany, 2012.) A retrospective cohort surveillance study was performed that included 167 non-albicans Candida The novel DNA microarray platform was developed by the Medical Mycology Research Center at Chiba University (MMRCChiba) to identify the following 12 genera and 32 fungal species, including 9 non-albicans Candida spp.: C. guilliermondii, C. lusitaniae, C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, C. dubliniensis, C. famata, C. kefyr, C. albicans For DNA microarray platform fabrication, the oligonucleotide probes used, consisting of 14 to 20 species-specific nucleotide sequences with biotin-labeled poly(T) anchors at the end of each nucleotide (Invitrogen, Showajima, Japan), were designed based on the internal transcribed spacer (ITS) sequences (ITS1 and ITS2) of the type strains (GenBank database, ATCC, Centraalbureau voor Schimmelcultures [CBS], and MMRC-Chiba). Multiple-sequence alignments were performed with the BioEdit software (version 7.1.3; http://www.mbio.ncsu.edu/BioEdit/bioedit .html). Conserved regions were also used as targets for genusspecific probes or as controls. Four to 11 oligonucleotide probe sequences were included for each Candida sp.; six probes were designed for common sequences, and two were designed for biotin l...

Fusarium napiforme systemic infection: case report with molecular characterization and antifungal susceptibility tests

“…The DNA microarray was performed as described by (Ferrari et al 2013 ), the oligonucleotide probes, consisting of 14 to 20 species-specific nucleotide sequences with biotin-labeled poly T anchors at the end of each nucleotide (Invitrogen, Showajima, Japan), were designed based on ITS1 and ITS2 sequences of the Type strains [GenBank database, American Type Culture Collection (ATCC), Centraalbureau voor Schimmelcultures (CBS) and MMRC-Chiba (IFM)]. Multiple-sequence alignments were performed using the BioEdit software (version 7.1.3.…”

Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

“…The sequence was 100% identical to C . tropicalis isolate 287 (Genbank Acc.# KU950724) [ 49 ]. An isolate previously identified as D .…”

Visible DNA Microarray System as an Adjunctive Molecular Test in Identification of Pathogenic Fungi Directly from a Blood Culture Bottle

“…DNA microarray platforms are shown to be highly sensitive and specific in studies that include fungal identification from fungi grown in culture media (24,26,27), from positive blood cultures (35)(36)(37), and from different clinical specimens, such as bronchial alveolar lavage fluid samples and blood samples (38,39). Limitations that could impair the microarray sensitivity would be the presence of a low number of fungal cells in the clinical sample and inefficient DNA extraction.…”

“…This platform was based on a panel of oligonucleotide probes that were designed on the basis of internal transcribed spacer (ITS) regions (24) of the rRNA gene to identify a variety of fungal species simultaneously in one polycarbonate slide, for which the results were visible to the naked eye (25). This array was previously studied by testing fungal DNA extracted from isolates grown in culture medium plates (25)(26)(27).…”