SHORT REPORT

Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections

P J Paterson¹, S Seaton¹, J McLaughlin², C C Kibbler¹

¹ Department of Medical Microbiology, Royal Free Hospital, Pond Street, London NW3 2QG, UK
² Department of Pathology, Royal Free Hospital

Correspondence to:
Correspondence to:
Dr C C Kibbler
Department of Medical Microbiology, Royal Free Hospital, Pond Street, London NW3 2QG, UK; kibbler{at}rfc.ucl.ac.uk

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^TM 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.

Keywords: aspergillus; tissue; DNA extraction; polymerase chain reaction

Abbreviations: IFI, invasive fungal infection; PCR, polymerase chain reaction

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This article has been cited by other articles:

• Paterson, P J, Seaton, S, Yeghen, T, McHugh, T D, McLaughlin, J, Hoffbrand, A V, Kibbler, C C (2005). Molecular confirmation of invasive infection caused by Chaetomium globosum. J. Clin. Pathol. 58: 334-334 [Full Text]