title: PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue.
abstract: BACKGROUND
  Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities.
  
  
  OBJECTIVE
  To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples.
  
  
  METHODS
  DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes.
  
  
  RESULTS
  Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases.
  
  
  CONCLUSIONS
  The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.
authors.affiliation: Institute for Tropical Medicine, University Hospital Tübingen, Keplerstrasse 15, 72074 Tübingen, Germany. ralf.bialek@med.uni-tuebingen.de
authors.forename: R
authors.initials: R
authors.lastname: Bialek
authors.forename: F
authors.initials: F
authors.lastname: Konrad
authors.forename: J
authors.initials: J
authors.lastname: Kern
authors.forename: C
authors.initials: C
authors.lastname: Aepinus
authors.forename: L
authors.initials: L
authors.lastname: Cecenas
authors.forename: G M
authors.initials: GM
authors.lastname: Gonzalez
authors.forename: G
authors.initials: G
authors.lastname: Just-Nübling
authors.forename: B
authors.initials: B
authors.lastname: Willinger
authors.forename: E
authors.initials: E
authors.lastname: Presterl
authors.forename: C
authors.initials: C
authors.lastname: Lass-Flörl
authors.forename: V
authors.initials: V
authors.lastname: Rickerts
mesh_terms: D001228:Aspergillosis / Q000175:diagnosis / Q000382:microbiology*; D001230:Aspergillus / Q000145:classification / Q000302:isolation & purification; D001483:Base Sequence; D004271:DNA, Fungal / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D009090:Mucorales / Q000145:classification / Q000302:isolation & purification; D009091:Mucormycosis / Q000175:diagnosis / Q000382:microbiology*; D016533:Mycological Typing Techniques / Q000379:methods; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012331:RNA, Fungal / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D004271:DNA, Fungal; D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S