title: Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy.
abstract: Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by tissue fixation. We used realtime PCR to quantify human and fungal DNA from formalin-fixed, paraffin-embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing the incubation temperature from 65°C to 90°C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy.
authors.affiliation: Robert Koch Institut, Berlin, Germany. RickertsV@rki.de
authors.forename: Volker
authors.initials: V
authors.lastname: Rickerts
authors.forename: Prasanna D
authors.initials: PD
authors.lastname: Khot
authors.forename: Daisy L
authors.initials: DL
authors.lastname: Ko
authors.forename: David N
authors.initials: DN
authors.lastname: Fredricks
mesh_terms: D001228:Aspergillosis / Q000382:microbiology; D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification; D002175:Candida / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000302:isolation & purification*; D005557:Formaldehyde / Q000737:chemistry*; D015894:Genome, Human; D006358:Hot Temperature; D006801:Humans; D008168:Lung / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods
chemical_list: D004271:DNA, Fungal; D005557:Formaldehyde