title: Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases.
abstract: 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.
authors.affiliation: Università Vita-Salute San Raffaele, Facoltà di Medicina e Chirurgia, Milan, Italy.
authors.forename: Nicasio
authors.initials: N
authors.lastname: Mancini
authors.affiliation: Università Vita-Salute San Raffaele, Facoltà di Medicina e Chirurgia, Milan, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Perotti
authors.forename: Cristina M
authors.initials: CM
authors.lastname: Ossi
authors.forename: Annalisa
authors.initials: A
authors.lastname: Cavallero
authors.forename: Stanislav
authors.initials: S
authors.lastname: Matuška
authors.forename: Giorgio
authors.initials: G
authors.lastname: Paganoni
authors.affiliation: Università Vita-Salute San Raffaele, Facoltà di Medicina e Chirurgia, Milan, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Burioni
authors.forename: Paolo
authors.initials: P
authors.lastname: Rama
authors.affiliation: Università Vita-Salute San Raffaele, Facoltà di Medicina e Chirurgia, Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Clementi
mesh_terms: D001483:Base Sequence; D003315:Cornea / Q000382:microbiology; D003316:Corneal Diseases / Q000175:diagnosis / Q000382:microbiology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology*; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012340:RNA, Ribosomal, 5.8S / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D012340:RNA, Ribosomal, 5.8S