title: Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS [corrected] software for identification of filamentous fungi in a clinical laboratory.
abstract: Filamentous fungal infections have recently increased because of the increasing numbers of immunocompromised hosts. In this study, we evaluated DNA sequencing of the D1/D2 region of the large subunit of the 28S ribosomal RNA gene and the internal transcribed spacer (ITS) region using SmartGene (SG; SmartGene Inc., Raleigh, NC) for the identification of a broad range of commonly encountered filamentous fungi. The SG proofreaders were used to upload, align, and edit fragments, and the resultant sequences were interpreted using the quality-controlled SG database. The results were compared with reference identifications using conventional phenotypic methods or ITS DNA sequences obtained from GenBank if phenotypic identifications were inconclusive. A total of 146 clinical isolates were included in this study, representing 49 different genera. The overall agreements of the D1/D2 and the ITS sequencing methods to reference identification were 97.2% (95% CI, 93.1% to 98.9%) and 97.7% (95% CI, 92.8% to 99.4%), respectively. Of the 146 isolates, 18 (12.3%) did not amplify using the ITS universal primers after repeated attempts and, therefore, could not be sequenced using this target. Correct identification was achieved for 100% (95% CI, 97.4% to 100%) of the isolates when applying both the D1/D2 and ITS targets. In summary, DNA sequencing using SG software provides a rapid, accurate, and reliable tool for the identification of filamentous fungi in a clinical laboratory.
authors.affiliation: The Johns Hopkins Hospital Microbiology Laboratory, Department of Pathology, The Johns Hopkins University School of Medicine, 600 N Wolfe St, Baltimore, MD 21287, USA. nkwiatk1@jhmi.edu
authors.forename: Nicole P
authors.initials: NP
authors.lastname: Kwiatkowski
authors.forename: Wisal M
authors.initials: WM
authors.lastname: Babiker
authors.forename: William G
authors.initials: WG
authors.lastname: Merz
authors.forename: Karen C
authors.initials: KC
authors.lastname: Carroll
authors.forename: Sean X
authors.initials: SX
authors.lastname: Zhang
mesh_terms: D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005658:Fungi / Q000235:genetics*; D012339:RNA, Ribosomal, 28S / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D012339:RNA, Ribosomal, 28S