title: Detection of mucormycetes and other pathogenic fungi in formalin fixed paraffin embedded and fresh tissues using the extended region of 28S rDNA.
abstract: Molecular methods of detection based on DNA-sequencing of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) or 5΄ end region of 28S (D1-D2 region) of ribosomal RNA gene (rDNA) have been used extensively for molecular identification and detection of fungal infections. However, these regions are not always informative for identification of mucormycetes and other rare fungal pathogens as they often contain large introns, heterogenic regions, and/or cannot be PCR-amplified using broad range fungal PCR primers. In addition, because of the difficulties of recovering intact fungal DNA from human specimens, smaller regions of DNA are more useful for the direct detection of fungal DNA in tissues and fluids. In this study, we investigated the utility of 12F/13R PCR primers targeting a 200-230 bp region of the extended 28S region of rDNA for molecular identification of fungal DNA in formalin fixed paraffin embedded tissues and other clinical specimens. We demonstrated that this region can be successfully used for identification of all genera and some species of clinically relevant mucormycetes, as well as other medically important fungi, such as Aspergillus, Fusarium, Coccidioides, and Cryptococcus. We also demonstrated that PCR amplification and direct sequencing of the extended 28S region of rDNA was more sensitive compared to targeting the ITS2 region, as we were able to detect and identify mucormycetes and other fungal pathogens in tissues from patients with histopathological and/or culture evidence of fungal infections that were negative with PCR using ITS-specific primers.
authors.affiliation: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA.
authors.forename: Lalitha
authors.initials: L
authors.lastname: Gade
authors.affiliation: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA.
authors.forename: Steven
authors.initials: S
authors.lastname: Hurst
authors.affiliation: Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA.
authors.forename: S Arunmozhi
authors.initials: SA
authors.lastname: Balajee
authors.affiliation: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA.
authors.forename: Shawn R
authors.initials: SR
authors.lastname: Lockhart
authors.affiliation: Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA.
authors.forename: Anastasia P
authors.initials: AP
authors.lastname: Litvintseva
mesh_terms: D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D005404:Fixatives; D005557:Formaldehyde; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009181:Mycoses / Q000175:diagnosis*; D010232:Paraffin; D057089:Pathology, Molecular / Q000379:methods*; D016133:Polymerase Chain Reaction; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D005404:Fixatives; D012339:RNA, Ribosomal, 28S; D005557:Formaldehyde; D010232:Paraffin
keywords: 28S; FFPE; mucorales; mucormycetes; rDNA