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

Abstract

A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one , two, and one ). Of note, the microarray detected DNA in two blood cultures from the same patient, whereas the automated system was only positive for Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.

<p>Current Aspects in the Biology, Pathogeny, and Treatment of <em>Candida krusei</em>, a Neglected Fungal Pathogen</p>

“…Based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene a chip to identify 32 fungal pathogens was recently reported. 162 Even though the results of the microarray and the automated system Vitek 2 were concordant in 96.7% of cases for all the pathogens tested, for the case of C. krusei the specificity was of 100%. 162 The multianalyte profiling system has also been adapted for the identification of Candida species.…”

“…162 Even though the results of the microarray and the automated system Vitek 2 were concordant in 96.7% of cases for all the pathogens tested, for the case of C. krusei the specificity was of 100%. 162 The multianalyte profiling system has also been adapted for the identification of Candida species. This consists of mixed polystyrene beads covalently linked to specific DNA probes for C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, and C. dubliniensis, which are incubated with amplicons containing the ITS2 region of Candida species rRNA gene previously amplified with universal fungal primers.…”

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis

“…Several studies with array platforms were designed to identify one or more fungal species in the same assay (13,18,30,31). The insertion of oligonucleotide sequences for the detection of various genera and fungi species in a single platform reduces the operational cost and allows the identification of several species of fungi in the same reaction and the observation of cross reactions.…”

“…A reliable identification of Cryptococcus species may facilitate the proper antifungal treatment and improve clinical and epidemiological surveillance strategies. Several studies have been published on the application of molecular techniques for the identification and diagnosis of Cryptococcus , but there is still a need for new studies and technologies to implement the diagnosis of this mycosis ( 10 – 13 ).…”

Biosensors and Diagnostics for Fungal Detection

“…These ribosomal genes exist in close proximity in the genome and are separated by internal transcribed regions (ITS1 and ITS2) which differ between fungal species and can be exploited to achieve an identification at the species level. These regions have therefore proved very fruitful in developing tests to identify fungi isolated from clinical samples [ 90 ]. These PCR tests are highly sensitive, so care must be taken when being used for direct diagnostic purposes to consider possible confounding positives originating from colonizing or contaminating organisms in patient samples [ 91 ].…”