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Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections
  1. P J Paterson1,
  2. S Seaton1,
  3. J McLaughlin2,
  4. C C Kibbler1
  1. 1Department of Medical Microbiology, Royal Free Hospital, Pond Street, London NW3 2QG, UK
  2. 2Department of Pathology, Royal Free Hospital
  1. Correspondence to:
 Dr C C Kibbler
 Department of Medical Microbiology, Royal Free Hospital, Pond Street, London NW3 2QG, UK; kibblerrfc.ucl.ac.uk

Abstract

Background/Aims: Invasive infection with emerging moulds is increasing in incidence and reliable methods for speciating these organisms in tissue sections need to be developed.

Methods: Two methods for extracting fungal DNA from paraffin wax embedded tissue sections, based on the TaKaRa DEXPAT kit and QIAamp® DNA mini kit, were optimised and compared. DNA was amplified by PCR using pan-fungal probes, and detected by Southern blot hybridisation using a high stringency method with a probe specific for Aspergillus fumigatus and A flavus.

Results: The method based on the TaKaRa DEXPAT kit, with additional steps using lyticase and ethanol precipitation, was superior. Less than 10 conidia were detectable using spiked samples and a positive result was obtained with 100% of clinical samples known to be culture positive for A fumigatus. Other moulds could be identified by using species specific probes or by sequencing PCR products.

Conclusions: The method based on the TaKaRa DEXPAT kit could detect less than 10 conidia/sample. The method allowed accurate identification of A fumigatus and A flavus and other species could be identified using species specific probes or by DNA sequencing. These methods will provide a valuable diagnostic tool for both patient management and future antifungal and epidemiological studies.

  • aspergillus
  • tissue
  • DNA extraction
  • polymerase chain reaction
  • IFI, invasive fungal infection
  • PCR, polymerase chain reaction
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Footnotes

  • This work was undertaken by Dr P J Paterson, Mrs S Seaton, Dr J McLaughlin, and Dr C C Kibbler with the Royal Free Hampstead NHS Trust who received a proportion of its funding from the NHS Executive; the views expressed in this publication are those of the authors and not necessarily those of the Trust or the NHS Executive.

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