ABSTRACT
The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.
Subject(s)
Fungi/isolation & purification , Mycoses/diagnosis , Polymerase Chain Reaction/methods , Antifungal Agents , Aspergillus/drug effects , Candida/drug effects , DNA Probes , DNA, Fungal/genetics , DNA, Intergenic , DNA, Ribosomal/genetics , DNA, Ribosomal Spacer/genetics , Drug Resistance, Fungal , Fungi/genetics , Humans , Mycoses/microbiology , Sensitivity and Specificity , Taq PolymeraseABSTRACT
An increase in the incidence of fungal infections has highlighted the need for rapid and precise detection and identification methods in clinical mycology. This report describes the data obtained on corneal samples from 24 patients with suspected keratomycosis using a conventional cultural approach in parallel with PCR amplification and sequencing of the internal transcribed spacers (ITSs) of the rDNA regions. Using the cultural approach, seven samples (58.3 % of the 12 samples positive for an infectious pathogen) tested positive for a fungal aetiology, with final identification taking a mean time of more than 5 days. In two cases, diagnosis required 10 days. Using the ITS-based molecular approach, a direct diagnosis was obtained in only five of the seven fungus-positive cases (71.4 %) starting from the clinical samples, but identification was still possible in all seven cases within 24 h (by using 16 h cultures for the two remaining cases). Despite the less-than-optimal sensitivity when working directly on clinical samples, the obtained data indicate that the molecular strategy used in this study is a useful complement to the conventional diagnostic approaches used for keratomycosis and, in particular, allows precise and fast fungal identification, in response to the clinical requirements. Similar studies on larger panels of patients and on different clinical samples are required for further investigation of the clinical potential of ITS-based approaches in the diagnosis of mycotic infections.
Subject(s)
Corneal Diseases/microbiology , Eye Infections, Fungal/microbiology , Fungi/isolation & purification , Polymerase Chain Reaction/methods , Base Sequence , Cornea/microbiology , Corneal Diseases/diagnosis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Eye Infections, Fungal/diagnosis , Fungi/genetics , Humans , Molecular Sequence Data , RNA, Ribosomal, 5.8S/chemistry , RNA, Ribosomal, 5.8S/genetics , Sequence Alignment , Sequence Analysis, DNAABSTRACT
The present report describes the diagnostic strategy followed in a case of keratomycosis. Together with conventional methods, a molecular strategy that involved the direct sequencing of an amplified portion of the genome encompassing the internal transcribed spacer 1 and 2 regions and sequence analysis was used. The data highlight the diagnostic role of molecular techniques, in parallel with conventional methods, in the management of ocular infections of fungal aetiology.
