title: Detection of tropical fungi in formalin-fixed, paraffin-embedded tissue: still an indication for microscopy in times of sequence-based diagnosis?
abstract: BACKGROUND
  The aim of the study was the evaluation of panfungal PCR protocols with subsequent sequence analysis for the diagnostic identification of invasive mycoses in formalin-fixed, paraffin-embedded tissue samples with rare tropical mycoses.
  
  
  METHODS
  Five different previously described panfungal PCR/sequencing protocols targeting 18S and 28S ribosomal RNA gene fragments as well as internal transcribed spacer 1 and 2 fragments were evaluated with a collection of 17 formalin-fixed, paraffin-embedded tissue samples of patients with rare and/or tropical invasive mycoses, comprising chromoblastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, mycetoma/maduromycosis, and rhinosporidiosis, in a proof-of-principle analysis.
  
  
  RESULTS
  The primers of the panfungal PCRs readily and predominantly reacted with contaminating environmental fungi that had deposited on the paraffin blocks. Altogether three sequence results of histoplasmosis and mycetoma samples that matched the histological assessment were associated with sample age <10 years and virtually without PCR inhibition.
  
  
  CONCLUSIONS
  The high risk of amplifying environmental contaminants severely reduces the usefulness of the assessed panfungal PCR/sequencing protocols for the identification of rare and/or tropical mycoses in stored formalin-fixed, paraffin-embedded tissues. Histological assessment remains valuable for such indications if cultural differentiation is impossible from inactivated sample material.
authors.affiliation: Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Bernhard-Nocht Street 74, 20359 Hamburg, Germany ; Institute for Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, 18057 Rostock, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: Frickmann
authors.affiliation: Central Laboratory Department/Department of Clinical Chemistry, University Medicine Goettingen, Robert Koch Street 40, 37075 Göttingen, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Loderstaedt
authors.affiliation: Department of Infectious Disease Pathology, Bernhard Nocht Institute for Tropical Medicine Hamburg, Bernhard Nocht Street 74, 20359 Hamburg, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Racz
authors.affiliation: Department of Infectious Disease Pathology, Bernhard Nocht Institute for Tropical Medicine Hamburg, Bernhard Nocht Street 74, 20359 Hamburg, Germany.
authors.forename: Klara
authors.initials: K
authors.lastname: Tenner-Racz
authors.affiliation: Department of Infectious Disease Pathology, Bernhard Nocht Institute for Tropical Medicine Hamburg, Bernhard Nocht Street 74, 20359 Hamburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Eggert
authors.affiliation: Department of Infectious Disease Pathology, Bernhard Nocht Institute for Tropical Medicine Hamburg, Bernhard Nocht Street 74, 20359 Hamburg, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Haeupler
authors.affiliation: LADR GmbH MVZ Dr. Kramer & Kollegen, Lauenburger Straße 67, 21502 Geesthacht, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bialek
authors.affiliation: Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Bernhard-Nocht Street 74, 20359 Hamburg, Germany.
authors.forename: Ralf Matthias
authors.initials: RM
authors.lastname: Hagen
mesh_terms: D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005557:Formaldehyde; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D009181:Mycoses / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology; D016612:Paraffin Embedding; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000302:isolation & purification*; D012339:RNA, Ribosomal, 28S / Q000235:genetics / Q000302:isolation & purification*; D016707:Tissue Fixation
chemical_list: D004271:DNA, Fungal; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D005557:Formaldehyde