title: Don't throw your blood clots away: use of blood clot may improve sensitivity of PCR diagnosis in invasive aspergillosis.
abstract: BACKGROUND
  The diagnosis of invasive aspergillosis (IA) remains challenging and frequently is not made until after death. Histopathological examination remains central to confirmation of diagnosis but often requires invasive procedures to obtain tissue for the examination. Detection of aspergillus DNA by quantitative PCR (qPCR) offers the potential for earlier diagnosis due to higher sensitivity, but PCR in clinical use is poorly reproducible, with different centres reporting variable results and often using different extraction and analytical methods.
  
  
  OBJECTIVE
  To optimise the performance of aspergillus PCR as a diagnostic modality.
  
  
  METHODS
  A rat inhalation model of invasive aspergillosis was used to optimise the methodology of diagnostic aspergillus PCR. Infected animals were terminally bled at 4 days post-infection; samples of EDTA blood, serum and the residual clot were pooled for subsequent analysis. DNA was extracted from each fraction using a variety of methods and an optimised qPCR reaction using an Aspergillus fumigatus primer set performed.
  
  
  RESULTS
  Significantly more aspergillus DNA was detected from the clot than EDTA and serum samples. Enzymatic and mechanical pretreatment reduced the yield of fungal DNA. There was some evidence that the average Ct values were greater for the EZ1 BioRobot than the MagNA Pure automated extractor, but this did not reach statistical significance at the 5% level (p = 0.078).
  
  
  CONCLUSIONS
  Automated extraction from the clot present in a blood sample will increase DNA yield and improve the diagnostic sensitivity of the test.
authors.affiliation: Microbiology Department, Royal Hospital for Sick Children, Glasgow, UK.
authors.forename: E
authors.initials: E
authors.lastname: McCulloch
authors.forename: G
authors.initials: G
authors.lastname: Ramage
authors.forename: B
authors.initials: B
authors.lastname: Jones
authors.forename: P
authors.initials: P
authors.lastname: Warn
authors.forename: W R
authors.initials: WR
authors.lastname: Kirkpatrick
authors.forename: T F
authors.initials: TF
authors.lastname: Patterson
authors.forename: C
authors.initials: C
authors.lastname: Williams
mesh_terms: D000818:Animals; D000925:Anticoagulants; D001228:Aspergillosis / Q000175:diagnosis*; D001232:Aspergillus fumigatus / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000097:blood*; D004492:Edetic Acid; D023421:Models, Animal; D008969:Molecular Sequence Data; D016533:Mycological Typing Techniques; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013927:Thrombosis / Q000382:microbiology*
chemical_list: D000925:Anticoagulants; D017931:DNA Primers; D004271:DNA, Fungal; D004492:Edetic Acid