Author:

P J Paterson, S Seaton, J McLaughlin and C C Kibbler

Date: 6 January 2004

Abstract:

Background/Aims: Invasive infection with emerging mouldsis increasing in incidence and reliable methods for speciatingthese organisms in tissue sections need to be developed.Methods: Two methods for extracting fungal DNA fromparaffin wax embedded tissue sections, based on the TaKaRaDEXPATTM kit and QIAampH DNA mini kit, were optimisedand compared. DNA was amplified by PCR using pan-fungalprobes, and detected by Southern blot hybridisation using ahigh stringency method with a probe specific for Aspergillusfumigatus and A flavus.Results: The method based on the TaKaRa DEXPAT kit, withadditional steps using lyticase and ethanol precipitation, wassuperior. Less than 10 conidia were detectable using spikedsamples and a positive result was obtained with 100% ofclinical samples known to be culture positive for A fumigatus.Other moulds could be identified by using species specificprobes or by sequencing PCR products.Conclusions: The method based on the TaKaRa DEXPAT kitcould detect less than 10 conidia/sample. The methodallowed accurate identification of A fumigatus and A flavusand other species could be identified using species specificprobes or by DNA sequencing. These methods will provide avaluable diagnostic tool for both patient management andfuture antifungal and epidemiological studies.

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