Evaluation of different molecular approaches for direct identification of fungal agents in clinical samples

M. Perotti, C.M. Ossi, M. Sampaolo, S. Carletti, M. Sassi, R. Burioni, M. Clementi and N. Mancini

Author address: 

Diagnostica e Ricerca San Raffaele; Universita` Vita-Salute San Raffaele, Laboratorio di Microbiologia, Milano, Italy

Abstract: 

The increasing diffusion of immunosoppression conditions and the widespread use of antimycotic drugs has led to a higher incidence of fungal infections. In addition to well-documented opportunists, new emerging agents are now causing of life-threatening infections in immunocompromised patients. Rapid and specific diagnosis represents a crucial element in the clinical management of these conditions. Firstly, standard microbiological tests are certainly necessary, but often lack rapidity usually taking longer than a week for growth and definitive identification of the aetiologic agent. Secondly, the identification of infectious agent is not just a mycological curiosity, but could be important for the choice of an effective therapy. Thirdly, a conventional mycological identification approach could be elusive in some cases, especially when morphological features are not easily differentiated. In this context, and in particular focusing our attention on the management of suspected fungal keratitis, we recurred to an already described molecular strategy including the direct sequencing of an amplified portion of the genome encompassing the highly divergent internal transcribed spacers 1 and 2 (ITS1-2). Along with classical cultural approaches, DNA extracted from corneal samples of 20 patients was amplified with a couple of universal fungal primers. Sequences of amplified products were submitted to BLAST database and the sequence with the highest bit score was considered for identification. The approach resulted absolutely concordant to culture reducing to only one day the time needed for identification and helping classical approach in one case of uncommon Microsporum canis keratomycosis in which identification was not straightforward. Moreover, in order to evaluate the reliability of this approach we performed a phylogenetic analysis of ITS1 and ITS2 sequences of reference strains of fungal isolates described in different ocular infections. A multiplesequence alignment and the construction of a phylogenetic tree were performed. This approach showed that ITS1 and ITS2 genetic divergences allow identification at the species level. Given this background, we devised a species-specific real-time PCR targeting fungal agents usually described in ocular infections. From sequences alignment, we designed molecular beacon probes recognizing, along with a ribosomal target conserved among fungi, ITS1-2- specific targets for fungal species commonly involved in keratomycosis (Aspergillus spp., Scedosporium apiospermum., Scedosporium prolificans, Fusarium spp., Candida spp). This approach presents some advantages if compared to sequence analysis of amplified ITS1 ITS2 regions, as reduced risk of false positives due to carryover, improved sensitivity and reproducibility and lowering of processing time. Moreover, the molecular specificity of this approach could also allow its use on other non-ocular samples even if contaminated by resident flora.
2005

abstract No: 

P140

Full conference title: 

2nd Trends in Medical Mycology
    • TIMM 2nd (2010)