DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients


Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Mörz H, Hehlmann R, Buchheidt D

Date: 11 September 2007


The increasing incidence of invasive fungal infections (IFI) in immunocompromised patients emphasizes the need to improve diagnostic tools. We established a DNA microarray to detect and identify DNA from 14 fungal pathogens (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, Candida dubliniensis, Candida glabrata, Candida lusitaniae, Candida tropicalis, Fusarium oxysporum, Fusarium solani, Mucor racemosus, Rhizopus microsporus, Scedosporium prolificans, Trichosporon asahii) in blood, bronchoalveolar lavage (BAL) and tissue samples from high risk patients. The assay combines multiplex polymerase chain reaction (PCR) and consecutive DNA microarray hybridization. PCR primers and capture probes were derived from unique sequences of the 18S, 5.8S and the internal transcribed spacer 1 (ITS 1) regions of the fungal rRNA genes. Hybridization with genomic DNA of fungal species resulted in species-specific hybridization patterns. Testing clinical samples from 46 neutropenic patients with proven, probable and possible IFI or without IFI, A. flavus, A. fumigatus, C. albicans, C. dubliniensis, C. glabrata, F. oxysporum, F. solani, R. microsporus, S. prolificans and T. asahii were detected. In 22/22 patients (without IFI, n=5; possible IFI, n=17), negative diagnostic results corresponded with negative microarray data. From 11 patients with proven (n=4), probable (n=2) and possible IFI (n=5) positive microarray data were validated by other diagnostic findings. In 11/11 patients with possible IFI the microarray results provided additional information. In 2 patients with proven respectively probable invasive aspergillosis, microarray results were negative. The assay detected genomic DNA from 14 fungal pathogens out of clinical samples, pointing to a high significance for improving diagnosis of IFI.

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