A Simplified Protocol for Rapid Sequence-Based Fungal Identification from Culture or Formalin-Fixed, Paraffin Embedded (FFPE) Tissues
David E Nowak, Dhananjay A Chitale, Robert Tibbetts. Henry Ford Hospital, Detroit, MI
Background: Identification of fungi is often a challenge in clinical microbiology, especially in small samples. Routine culture often results in unidentifiable sterile hyphae or no growth and histologic evaluation is hampered by variable tissue morphotypes. We developed a sequence-based assay using available reagents, kits and equipment familiar to any molecular pathology laboratory. The strengths of this approach include: simplicity, definitive results, and species-specific data in less than 12 hours.
Design: Culture fungi were selected from in-house ATCC stocks and sterile hyphae cultured fungi. FFPE specimens for analysis were selected randomly based on tissue source from archival tissues retrieved from pathology files. Prior diagnoses were blinded. DNA was extracted (QIAamp mini kit, Qiagen) and amplified using published primers ITS3 and ITS4, hybridizing to conserved sequences between the 5s and 28s rRNA genes of all fungi. DNA was purified (QIAquick PCR cleanup kit, Qiagen), sequenced (BigDye3.1, ABI) and used to interrogate the NCBI BLAST nucleotide-to-nucleotide database. Species identification was based on best sequence alignment.
Results: DNA was successfully amplified from cultures and FFPE tissues. ATCC isolates of Cryptococcus laurentii, Candida glabrata, Candida parapsilosis, and Candida tropicalis were all verified: 99%, (gb FJ153214.1); 99%, (gb CR380958.2), 100%, (gb HQ263346.1), 98%, (gb JF300164.1) match, respectively. Sterile hyphae from four cases identified: Ajellomyces dermatitidis, 99%, (gb EF592163.1); Pichia kudriavzevii, 98%, (gb HM771638.1); Cladosporium cladosporioides, 99%, (gb JN253511.1); and Coprinellus micaceous, 98%, (gb GU227721.1) match, respectively. Trichophyton rubrum, Cladosporium cladosporioides, and Cladosporium tenuissimum were identified in toe nail with 96% (gb FM178326.1); 96% (gb JN253511.1); 89% (gb Y15966.1) match, respectively. In lung, a tissue diagnosed as Cryptococcus sp.was identified as Ajellomyces dermatitidis by sequencing with 99% match (GB EF592163.1). Candida sp. with previous histological diagnosis in lung, skin, and esophagus identified as Candida albicans with 93%, (gb HQ014713.1); 99%, (gb HQ014713.1); 96%, (gb HQ014723.1) match, respectively.
Conclusions: We have established an assay for fungal identification based on DNA sequence that is both faster and more definitive than culture or tissue morphology with a unified workflow for fresh, frozen or FFPE fungi in less than 12 hours.
Monday, March 19, 2012 11:00 AM
Platform Session: Section H, Monday Morning