[Show abstract][Hide abstract] ABSTRACT: We developed a reverse line blot (RLB) hybridization-, and rolling circle amplification (RCA)-based assays for the identification of Trichoporon species and evaluated them with 48 isolates that had been previously recognized as belonging to eight species (Trichosporon asahii, T. cutaneum, T. dermatis, T. domesticum, T. inkin, T. japonicum, T. jirovecii, and T. laibachii). Results were compared to those obtained with DNA sequencing of three rRNA gene loci, i.e., the internal transcribed spacer (ITS) region, D1/D2 domain of the 28S rRNA gene and intergenic spacer 1 (IGS1) region. Using species-specific, or group-specific probes targeted at the ITS region and the D1/D2 domain, the RLB assay permitted accurate species identification of all 48 isolates with 100% specificity. Species-specific RLB probes correctly assigned 45/48 (94%) of the isolates (six species) with the exception of T. dermatis and T. japonicum isolates which were not targeted by the assay. Identification of T. dermatis relied on a positive hybridization result with the group-specific probe hybridizing with T. dermatis and T. jirovecii and the absence of a signal with the T. jirovecii-specific probe. T. japonicum strains were first assigned to the T. asahii-T. japonicum group by hybridization with the two species group-specific probe and then as T. japonicum by the absence of signal with a T. asahii-specific probe. Twelve species-specific RCA probes targeting the eight species studied detected templates of all 48 Trichosporon isolates and an artificial template of T. asteroides, all with good specificity. Both RLB and RCA are potential alternatives to DNA sequencing for the identification of Trichosporon species. The RLB approach is suited for the batched simultaneous analysis of large numbers of isolates, while RCA is more appropriate for the immediate study of single isolates. Comparative costs are US$7 and US$2 per assay for the RLB and RCA methods, respectively.
Medical mycology: official publication of the International Society for Human and Animal Mycology 11/2012; 51(3). DOI:10.3109/13693786.2012.723223 · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three reference and 45 clinical isolates of Trichosporon were analyzed by conventional phenotypic and molecular methods to determine the species and genotypes of Trichosporon isolates from China. Target loci for molecular methods included the internal transcribed spacer (ITS) region, the D1/D2 domain of the 26S rRNA gene, and the intergenic spacer 1 (IGS1) region. Identification of eight Trichosporon species was achieved, of which Trichosporon asahii was the most common. Of the sequence-based molecular methods, the one targeting the D1/D2 domain assigned 97.9% (47/48) of isolates (seven species) correctly, while tests targeting both the ITS and IGS1 regions correctly identified all 48 isolates. The commercial API 20C AUX and Vitek 2 Compact YST systems correctly identified 91.9% and 73% of isolates when their biochemical profiles were queried against those of species contained in the databases, respectively, and misidentified 63.6% and 36.4% of isolates of species that were unclaimed by the databases, respectively. The predominant genotype among T. asahii clinical isolates, genotype 4 (51.4%), is rarely found in other countries. Voriconazole and itraconazole were the most active drugs in vitro against all the Trichosporon species tested, while caspofungin and amphotericin B demonstrated poor activity.