Development of a DNA Microarray for Detection and Identification of Fungal Pathogens Involved in Invasive Mycoses

Abstract

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is timeconsuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS1 and ITS4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 precharacterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.

Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases

“…Nonetheless, in the two discordant cases, definitive identification was possible from colonies before they could be assessed by classical identification. Of note, the present study indicates that the high specificity of the ITS regions, previously described only in studies performed on cultured isolates (Leinberger et al, 2005;Pryce et al, 2003), makes them suitable for identification of fungal pathogens directly from clinical samples; indeed, no false-positive reactions related to cross-amplification of human DNA or DNA derived from other sources (shown by the absence of amplified product in the five samples that were positive for non-fungal pathogens) were observed. These features, along with their potential use on automated supports, make ITS sequences valuable targets for setting up molecular approaches aimed at direct and rapid diagnosis of fungal pathogens at the species level, and could also be applied to non-ocular samples that are heavily contaminated by resident flora, as well as to samples from sterile sites.…”

“…The internal transcribed spacer (ITS) 1 and ITS2 rDNA sequences offer several advantages in this perspective (Iwen et al, 2002;Leinberger et al, 2005;Pryce et al, 2003). The ITS regions are non-coding sequences interspaced among highly conserved fungal rDNA and have been shown to have a high heterogeneity among different fungal genera and species (Iwen et al, 2002;Pryce et al, 2003).…”

DNA Microarray-Based Detection and Identification of Fungal Pathogens in Clinical Samples from Neutropenic Patients

“…For our assay, sensitive and specific detection and identification of the fungal pathogens were achieved by designing primer pairs complementary to the highly conserved 18S and 5.8S regions of the fungal rRNA genes and oligonucleotide capture probes complementary to the more variable ITS1 regions which enabled a differentiation of fungal species (13,14,15,16,20). As part of the rRNA genes, the ITS1 regions were chosen as targets because they are present in numerous copies in the fungal genome.…”

Development of a DNA Array for the Simple Identification of Major Filamentous Fungi in the Beverage Manufacturing Environment

“…Furthermore, almost all of the developed DNA arraybased evaluation methods detect spots with microscopic size after fluorescent labeling, which require specialized equipment for reading the hybridization results Leinberger et al, 2005;Spiess et al, 2007;Sato et al, 2010;Cao et al, 2011 . In the DNA array method developed in this study, hybridization results can be evaluated by the naked eye without the use of the expensive and specialized equipment. The present DNA array method is applicable to the on-site identification of fungi in soft drink manufacturing plants.…”

“…Only a few methods have been developed for targeting multiple species Leinberger et al, 2005;Chiang et al, 2006;Spiess et al, 2007;Cao et al, 2011 . While the methods had been reported for the identification of large numbers of microbial species and for the identification of fungi, these studies targeted medically important fungi Hsiao et al, 2005;Leinberger et al, 2005;Spiess et al, 2007;Sato et al, 2010;Sakai et al, 2014 . The development of the present DNA array method is the world s first attempt for identification of filamentous fungi in the soft drink and food manufacturing environments.…”