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

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.

Figure 1

Comparison of methods with spiked samples. (A) Gel of QIAamp method with and without lyticase. (B) Gel of TaKaRa method plus ethanol precipitation, with and without lyticase. (C) Blot of QIAamp methods. (D) Blot of TaKaRa methods. M, molecular weight marker; lanes 1–5, 10⁴, 10³, 10², 10¹, and 10⁰Aspergillus fumigatus conidia, respectively; lane 6, negative control. L, with lyticase.

Figure 2

Comparison of methods with clinical samples. (A) Gel of QIAamp method. (B) Gel of TaKaRa method. (C) Blot of QIAamp method. (D) Blot of TaKaRa method. M, molecular weight marker; lanes 1–5, clinical samples 1–5, respectively; lanes 6 and 7, negative controls.