Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections. Issue 6 (26th November 2003)

• Record Type: Journal Article
• Authors: Paterson, P J; Seaton, S; McLaughlin, J; Kibbler, C C
• Is Part Of: Journal of clinical pathology. Volume 56:Issue 6(2003)
• Page Start: 368
• Page End: 370
• Publication Date: 2003-11-26
• Subjects: aspergillus -- tissue -- DNA extraction -- polymerase chain reaction; IFI, invasive fungal infection -- PCR, polymerase chain reaction; Pathology -- Periodicals; Pathology, Molecular -- Periodicals; 616.0705
• DOI: 10.1136/mp.56.6.368
• Languages: English
• ISSNs: 0021-9746

Abstract

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.